Use of multivalent synthetic ligands of surface nucleolin for treating cancer or inflammation

ABSTRACT

A method for treating disorders involving deregulation of cell proliferation and/or angiogenesis comprising the administration of an effective amount of a multivalent synthetic compound comprising a support on which at least 3 pseudopeptide units are grafted, said compound being of formula (I).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/298,511 filed Oct. 24, 2008, which claims the benefit ofPCT/FR2007/000730, titled: Use of Multivalent Synthetic Ligands ofSurface Nucleolin for Treating Cancer or Inflammation, filed: 27 Apr.2007; which claims priority to French Patent Application FR 0603813,filed: 27 Apr. 2006.

INCORPORATION BY REFERENCE

In compliance with 37 C.F.R. §1.52(e)(5), an electronic CRF of thesequence listing is filed herewith: file name: D24262_Seqlisting_US_ST25.txt; size 17 KB; created on: Jan. 3, 2011; usingPatentIn-3.5, and Checker 4.4.0 is hereby incorporated by reference inits entirety. The data in the Computer Readable Form of the SequenceListing submitted herewith contains no new matter, and is fullysupported by the priority application, PCT/FR2007000730 filed 27 Apr.2007.

FIELD OF THE INVENTION

The present invention relates to the use of a multivalent syntheticcompound, which is significantly more resistant to at least one proteasethan a standard peptide bond, for preparing a medication intended forthe treatment of a diseases involving deregulation of cell proliferationand/or angiogenesis, and preferably acting as a surface nucleolinligand.

BACKGROUND

Cell division, or mitosis, is the process which allows cells to multiplyin order to repair or regenerate tissues and replace dead cells. Incancer cells, regulation of this process is defective and this is whythese cells divide anarchically and give rise to tumours. Thus, oneeffective therapeutic route to prevent the development of cancerconsists in blocking the division of cancerous cells using moleculeswith anti-mitotic properties.

Nevertheless, current anti-mitotic molecules (paclitaxel, better knownunder the name taxol, or colchicine for example) act without cellspecificity on all cells without distinction, thus causing many unwantedside effects. It is therefore essential to develop anti-mitoticmolecules with fewer harmful effects.

Every tumour needs nutrients and oxygen in order to grow. These elementsare provided by intratumoral blood vessels which result from a mechanismknown as angiogenesis. In fact, if these vessels are absent, tumourcells undergo a cell necrosis process, and tumour growth slows down thenstops. An example of another therapeutic route to combat cancertherefore consists in blocking the angiogenesis process by blocking themolecules controlling this mechanism.

The plurality of current anti-angiogenic molecules are specific to oneangiogenic factor. This monospecificity gives rise to resistancephenomena. Inhibition of one angiogenic factor type produces expressionof another type by angiogenic compensation mechanisms. It is thereforebeneficial to have available anti-angiogenic molecules with a broadspectrum of activity against the factors implicated.

However, inhibition of the angiogenesis process alone is generally foundto be insufficient to effectively block tumour growth. In addition, itdoes not block the formation of metastases.

It would therefore be extremely useful to have available new anti-cancermolecules capable of inhibiting both tumour cell proliferation and theangiogenesis process in the tumour. In fact, a recent study has shownthat a combination of two therapeutic molecules, one anti-mitotic andthe other anti-angiogenic, produces a synergetic effect andsignificantly increases the efficacy of overall treatment compared totreatment with only one of these molecules.

No molecule with both these effects, anti-mitotic and anti-angiogenic,has yet been reported.

Moreover, the majority of current anti-cancer agents are not trulyspecific to tumour cells and therefore also target healthy cells, thusgiving rise to many, and at times serious, side effects. This problemhas been resolved in some cases by the development of antibodies whichtarget the surface molecules of some tumours. However, the use ofantibodies poses other serious problems and the development of effectivetherapeutic antibodies that are nontoxic is a lengthy, uncertain andexpensive procedure. Moreover, the production of antibodies on a largescale and under strict health and safety conditions is particularlydifficult. As a result, treatments based on specific antibodies arestill far and few between and extremely costly.

Another problem linked to conventional anti-cancer drugs, such aspaclitaxel, is that these molecules are often highly hydrophobic whichmakes it necessary to develop complicated and expensive pharmaceuticalformulations in order to achieve acceptable bioavailability in vivo. Theproblem of in vivo bioavailability is all the more acute in the case oftreatment using nucleic acids since it is extremely difficult for themto reach their target cells in an efficacious and specific manner.

It would therefore be extremely useful to have available new anti-cancermolecules which present the following characteristics: much improvedefficacy as a result of their dual inhibitory action on tumourproliferation and angiogenesis such that they can be effective alone,without the use of conventional chemotherapy or radiotherapy and thusgreatly the limit side effects linked to these types of treatment, afairly broad spectrum of activity against angiogenic factors to preventresistance to treatment, very few side effects as a result of greaterspecificity towards tumour cells, a synthesis process that is easilyadaptable to an industrial scale, easier to use, notably as a result ofbetter bioavailability and/or longer half-life in vivo, in particular asa result of direct specificity for tumour cells, with good solubility inaqueous media and improved resistance to in vivo breakdown processes.

DESCRIPTION OF FIGURES

FIG. 1. A. Structure of nucleolin protein. Human nucleolin consists of707 amino acids. Nucleolin can be broken down into two main parts:(3,4): N-terminal (aa 1-308) and C-terminal (309-706). The N-terminaldomain consists of 4 long acid domains, consisting of an uninterruptedrepetition of glutamic acid and aspartic acid (A1, A2, A3, A4). TheC-terminal domain, consisting of alternating hydrophobic and hydrophilicregions forming 4 areas of binding to RNA called RBDs (for <<RNA BindingDomains>>: I, II, III, IV) and its extremity (aa 644-707) carries thehighly basic RGG domain comprised of Arg-Gly-Gly repetitions. B.Identification of the binding domain of compound HB19 to nucleolin: theRGG domain. Nucleolin constructions corresponding to the N- andC-terminal areas were obtained by in vitro transcription/translation ina system using rabbit reticulocyte lysates. Thus, whole nucleolin andthe N- and C-terminal parts contain amino acids 1-707, 1-308 and 309-707respectively labelled with [³⁵S] Met/Cys were produced. The labelledcrude product was then incubated with biotinylated HB19 and thecomplexes were purified on an avidine-agarose column. As expected, wholenucleolin interacts with HB19. On the other hand, the N-terminal part ofthe nucleolin rich in acid residues does not interact at all with thecompound whereas the C-terminal part of nucleolin contains the targetfor HB19 (14). Having identified that the C-terminal part of thenucleolin contains the target for HB19, various constructions (N° 1 to9) of this region were made up. The first construction corresponds tocDNA coding for the C-terminal part of human nucleolin including the 4RBDs and the RGG domain, in fusion with GST protein (GlutathioneS-Transferase) to allow detection with anti-GST antibodies. The otherconstructions, also in fusion with GST, correspond to this same part butone or more domains shorter. All these proteins are produced by E. coli.The capacity of HB19 to interact with each construction was tested byincubating crude bacterial extracts, expressing different nucleolinconstructions, with biotinylated HB19 which was then purified by fixingto Avidine-agarose. These samples were then analysed by polyacrylamidegel and GST, and revealed by immunodetection (Western Blot) usinganti-GST antibodies. The results show that the presence of the RGGdomain is necessary for the interaction between HB19 with the C-terminalpart of nucleolin. Moreover, the RGG domain alone is enough for thisinteraction.

FIG. 2. A. Structure of compound HB19. B. Structure of trivalentcompound Nucant 01 with a cyclic hexapeptide consisting of alternatingalanine residues (A) of configuration D and lysine residues (K) ofconfiguration L as the support. Three pseudopeptide units KΨPR (withΨ=CH₂—N) are covalently bound to the ε amino group of each of the lysineresidues. C. Structure of pentavalent compound Nucant 2 (SEQ ID NO :10)with a linear peptide as a support having a helicoidal structure ofsequence SEQ ID NO :8 in which 5 pseudopeptide units KΨPR (with Ψ=CH₂—N)are covalently bound to the ε amino group of each of the 5 lysineresidues, Ac represents a CH₃—CO— group. D. Structure pentavalentcompound Nucant 3 (SEQ ID NO :11) with a linear peptide as a supporthaving a helicoidal structure of sequence SEQ ID NO :9 in which 5pseudopeptide units KΨPR (with Ψ=CH₂—N) are covalently bound to the εamino group of each of the 5 lysine residues, Ac represents a CH₃—CO—group. E. Structure of hexavalent compound Nucant 6 (SEQ ID NO :16) witha linear peptide as a support having a helicoidal structure of sequenceSEQ ID NO :15 in which 5 pseudopeptide units KΨPR (with Ψ=CH₂—N) arecovalently bound to the ε amino group of each of the 6 lysine residues,Ac represents a CH₃—CO— group. F. Structure of hexavalent compoundNucant 7 (SEQ ID NO :17) with a linear peptide as a support having ahelicoidal structure of sequence SEQ ID NO :13 in which 6 pseudopeptideunits KΨPR (with Ψ=CH₂—N) are covalently bound to the ε amino group ofeach of the 6 lysine residues, Ac represents a CH₃—CO— group.

FIG. 3. Effect of A. anti-nucleolin (anti Nu), B. isotypical IgG, C.peptide F3, and D. HB-19 on the proliferation of NIH-3T3 cellsstimulated by HARP. Quiescent NIH-3T3 cells are stimulated or not by 4nM of HARP in the presence or not of HB19 at the concentrationsindicated. After 24 hours of incubation, cell proliferation wasdetermined by measuring the incorporation of tritiated thymidine. Theresults are given as a percentage with respect to the control stimulatedby HARP (100%). MSD (threshold), (**p<0.01 and ***p<0.001).

FIG. 4. Effect of pretreatment of NIH-3T3 cells with HB19 for one houron proliferation induced by HARP. NIH-3T3 cells were treated with HB19for one hour then washed and stimulated or not by 3.6 nM of HARP. After24 hours of incubation, cell proliferation was determined by measuringthe incorporation of tritiated thymidine. The results are given as apercentage with respect to the control stimulated by HARP (100%). MSD(threshold), (**p<0.01 and ***p<0.001).

FIG. 5. Effect of HB-19 on the proliferation of NIH-3T3 cells stimulatedby 0.2 nM FGF-2 (A) or by 5% foetal calf serum (B). Quiescent NIH-3T3cells are stimulated by FGF-2 or by 5% serum in the presence or not ofHB19 at the concentrations indicated. After 24 hours of incubation, cellproliferation was determined by measuring the incorporation of tritiatedthymidine. The results are given as a percentage with respect to thecontrol stimulated by HARP (100%). MSD (threshold), (**p<0.01 and***p<0.001).

FIG. 6. Effect of anti-nucleolin (anti Nu) (A), isotype IgG (B) andHB-19 (C) on MDA-MB231 growth on wet agar. MDA-MB231 cells are culturedin a culture medium with 0.35% agar on a 0.6% agar matrix. After 10 daysin culture, colonies with a diameter greater than 50 μm were counted. 5areas per well and each point in triplicate, (**p<0.01).

FIG. 7. Effect of anti-nucleolin (anti Nu) (A), and HB-19 (B) on B16-BL6growth on wet agar. B16-BL6 cells are cultured in a culture medium with0.35% agar on a 0.6% agar matrix. After 10 days in culture, colonieswith a diameter greater than 50 μm were counted. 5 areas per well andeach point in triplicate, (*p<0.05 and **p<0.01).

FIG. 8. Effect of HB-19 on angiogenesis. A. Effect of HB19 tested on invitro proliferation of HUVEC cells. 20000 HUVEC cells were cultured inwells and compound HB19 was added in different concentrations on eachday. Cells were counted after 6 days of treatment. B. Effect of HB19 (1μM) on the differentiation of HUVEC cells in a three-dimensionalcollagen gel cultured in the presence of HARP angiogenic factors (1 nM);VEGF (1 nM) and FGF-2 (3 nM) were also tested. After 4 days, tubularnetwork structures were counted. The results are presented in arbitraryunits. C. Effect of HB-19 on angiogenesis triggered HARP or FGF-2 in anin vivo angiogenesis model (/matrigel <<plug assay>>). Matrigel (300 μl)containing the indicated molecules is injected subcutaneously into mice.Mice were sacrificed after one week and matrigel was removed. 8 μm thickcuts were performed. After staining, the number of endothelial cells isestimated by image analysis. For each matrigel, 5 cuts and 4 mice wereanalysed per experimental point.

FIG. 9. Effect of HB-19 on tumour growth in an MDA-MB231 xenograftmodel. Human mammary carcinoma MDA-MB231 cells were injectedsubcutaneously athymic mice (nude). When the tumour reached a volume of200 mm³, mice were treated by subcutaneous route as shown in graph in A.B. Observation and measurement of tumours in mice sacrificed at day 40.C. Tumour weight in mice sacrificed at day 40.

FIG. 10. Effect of HB-19 on tumour growth in an MDA-MB231 xenograftmodel. Changes in tumour growth. Intraperitoneal (IP) or subcutaneous(SC) injections.

FIG. 11. Effect of HB-19 on metastatic tumour cells in a MDA-MB231xenograft model. Investigation of MDA-MB231 cells in the blood ofxenografted mice and treated or not with HB-19 was performed by fluxcytometry (FACS) using anti HLA-DR antibodies. HLA-DR⁺ are surroundedand the percentage of HLA-DR⁺ cells among blood cells is indicated. A.Blood of mice without an MDA-MB231 xenograft, untreated, percentage ofHLA-DR+ cells among peripheral blood cells: 0.36% B. Blood of micewithout an MDA-MB231 xenograft, untreated, percentage of HLA-DR+ cellsamong peripheral blood cells: 22.2% C. Blood of mice with MDA-MB231xenograft, treated with HB-19 by subcutaneous route, percentage ofHLA-DR+ cells among peripheral blood cells: 0.1% D. Blood of mice withan MDA-MB231 xenograft, treated with HB-19 by intraperitoneal route,percentage of HLA-DR+ cells among peripheral blood cells: 0.31%.

FIG. 12. Effect of HB-19 and Nucant 01 on the growth of NIH-3T3 cellsstimulated by HARP. Quiescent NIH-3T3 cells were stimulated or not by 4nM of HARP in the presence of HB19 or Nucant 01 at the concentrationsindicated. After 24 hours of incubation, cell proliferation wasdetermined by measuring the incorporation of tritiated thymidine. Theresults (mean of 3 points) are given as the percentage of cellproliferation with respect to the control stimulated by HARP in theabsence of HB19 and Nucant 01 (100% cell proliferation).

FIG. 13. Effect of HB-19, Nucant 2 and Nucant 3 on the growth of NIH-3T3cells stimulated by HARP. Quiescent NIH-3T3 cells were stimulated or notby 4 nM of HARP in the presence of HB19, Nucant 2 or Nucant 3 at theconcentrations indicated (0.1, 0.25 and 0.5>M). After 24 hours ofincubation, cell proliferation of NIH-3T3 cells was determined bymeasuring the incorporation of tritiated thymidine. The results (mean of3 points) are given as the percentage of cell proliferation with respectto the control stimulated by HARP (100% cell proliferation). The IC50concentrations (concentration leading to 50% inhibition of cellproliferation with respect to the control stimulated by HARP) are alsogiven.

FIG. 14. Effect of NUCANT 3, 6 and 7 on the proliferation of NIH-3T3cells stimulated by 5% FCS. NIH-3T3 cells are made quiescent by serumdeprivation are stimulated by 5% FCS in the presence or not of variousNUCANT 3, 6 and 7 concentrations ranging from 0.125 to 2 μM. After 24hours of incubation, cell proliferation was determined by measuring theincorporation of tritiated thymidine. The results are given as apercentage with respect to the control cells stimulated by 5% FCS. ID₅₀is indicated by the dotted line.

FIG. 15. Nucant 6 and Nucant 7 show better anti-surface nucleolinactivity than HB-19. ID₅₀: concentration in μM which inhibits surfacenucleolin by 50%. ID₉₅: concentration in μM which inhibits surfacenucleolin by 95%.

FIG. 16. Inhibitory effect of HB-19, Nucant 3, Nucant 6 and de Nucant 7on cell expression of nucleolin by MDA-MB 231 cells. MDA-MB 231 cellswere cultured in 75 cm² in DMEM containing 10% foetal calf serum. After2 days in culture, subconfluent cells (about 3×10⁶ cell per vial) weretreated with 10 μM of HB-19 (line 1), Nucant 3 (line 2), Nucant 6 (line3) or Nucant 7 (line 4) for 24 or 48 hours. Lines C represent untreatedcells. After 24 or 48 hours of treatment, cells were washed in PBS andincubated with 10 ml of DMEM containing 1% foetal calf serum andbiotinylated HB-19 (5 μM) for 45 minutes at room temperature. Afterintensive washing in PBS containing 1 mM EDTA (PBS-EDTA), cytoplasmicextracts were prepared using a lysis buffer containing 20 mM Tris HCl,pH 7.6, 150 mM NaCl, 5 mM MgCl₂, 0.2 mM phenylmethylsulfonyl fluoride, 5mM β-mercaptoethanol, aprotinin (1000 U/ml) and 0.5% Triton X-100. Thecomplex formed between surface nucleolin and biotinylated HB-19 wasisolated purification of the extracts using avidine-agarose (100 μl;ImmunoPure Immobilized Avidin, Pierce Chemical Company, USA) inPBS-EDTA. After 2 hours of incubation at 4° C., the avidine-agarosesamples were thoroughly washed with PBS-EDTA. These samples containingpurified surface nucleolin (A material corresponding to 2×10⁶ cells) andcrude cell extracts (B and C; material corresponding to 4×10⁵ cells)were denatured by heating in electrophoresis buffer containing SDS andanalysed by SDS-PAGE. The presence of surface nucleolin was revealed byimmunoblotting using D3 monoclonal antibodies (A and B). Electrophoresisanalysis after staining with Coomassie Blue is shown in C. Line Mcorresponds to molecular weight markers.

FIG. 17. Inhibition of angiogenesis in an ex vivo CAM model. 20 μl ofwater containing or not (control) HB-19 (10 μM; 0.6 μg) or Nucant 7 (10μM; 0.8 μg) are deposited on the surface of CAM. Observation of vesselsis carried out after 48 hours of incubation.

FIG. 18 Inhibition by HB-19 of the production of TNF-α by primary humanmononuclear peripheral blood cells (PBMC) stimulated by various LPSpreparations. PBMCs were isolated by centrifugation on a Ficoll densitygradient using whole human blood EDTA-potassium and resuspended in RPMI1640 containing 1% human serum AB (Invitrogen). Cells at a concentrationof 10⁶ cell/0.5 ml, in the absence (0) or presence (1 and 5 μM) ofHB-19, were stimulated with 100 ng/ml of LPS from Escherichia coli type0111: B4 and 055: B5, and LPS from Salmonella enterica serotype Re 595.The same PBMCs were stimulated with PMA: Ionomycin (Phorbol 12-myristate13-acetate: Ionomycin) at 20 ng/ml: 1 μM. PBMC cultures were incubatedat 37° C. in an incubator with 5% CO₂. Levels of TNF-α protein weremeasured by ELISA in culture supernatants collected after 20 hours ofincubation.

FIG. 19. Inhibition by HB-19 of the production of TNF-α and IL-6 byprimary peritoneal murine macrophages stimulated by various LPSpreparations. Peritoneal murine macrophages in the absence (−) orpresence (+) of 4 μM of HB-19 were either not stimulated (B4 0) orstimulated with LPS from Escherichia coli type 0111: B4 at 100 ng/ml (B4100) and 1000 ng/ml (B4 1000). Cell cultures were incubated at 37° C. inan incubator with 5% CO₂ for 20 hours and levels of TNF-α (A) and IL-6(B) were measured by ELISA.

FIG. 20 Inhibition by Nucant 7 of the production of TNF-α and IL-6 byprimary peritoneal murine macrophages stimulated by various LPS.Peritoneal murine macrophages in the absence (−) or presence (+) of 10μM of Nucant 7 were either not stimulated (−) or stimulated (+) with LPSfrom Escherichia coli type 0111: B4 at 10 ng/ml, 100 ng/ml and 1000ng/ml. Cell cultures were incubated at 37° C. in an incubator with 5%CO₂ for 20 hours and levels of TNF-α (A) and IL-6 (B) were measured byELISA.

FIG. 21 Inhibition by HB-19 of IL-8 production and ICAM-1 expression byhuman umbilical vascular endothelial cells (HUVEC) stimulated by LPS.HUVEC cells at 10 000 cells/cm² were cultured in 96-well plates in EBM-2medium containing 2% foetal calf serum. Cells in the absence or presenceof 5 μM of HB-19 were stimulated with Escherichia coli serotype 055: B5at 100 ng/ml. Cell cultures were incubated at 37° C. in an incubatorwith 5% CO₂ for 20 hours and IL-8 and ICAM-1 protein levels weremeasured by ELISA. HUVEC cells in the presence or absence of 5 μM ofHB-19 were used as the control for base levels.

FIG. 22 Inhibition by HB-19 of the production of TNF-α and IL-6 byprimary human mononuclear peripheral blood cells (PBMC) stimulated byStaphylococcus aureus bacteria inactivated by heat (HKSA, <<heat-killedStaphylococcus aureus>>). PBMCs were isolated by centrifugation on aFicoll density gradient using whole human blood EDTA-potassium andresuspended in RPMI 1640 containing 1% human serum AB (Invitrogen).Cells at a concentration of 10⁶ cell/0.5 ml, in the absence (control) orpresence (10 μM) of HB-19, Nucant 3, Nucant 6 or Nucant 7 orDexamethasone (Dex. 1 μg/ml), were stimulated with 10⁸ HKSA/ml particles(InvivoGen, San Diego, USA). PBMC cultures were incubated at 37° C. inan incubator with 5% CO₂ and TNF-α (A) and IL-6 (B) levels were measuredby ELISA in culture supernatants collected after 20 hours of incubation.

DESCRIPTION

The present invention relates to the use of a multivalent syntheticcompound comprising or consisting of a support on which at least 3pseudopeptide units are grafted, said compound being of formula (I):

where each X independently represents any amino acid; Y₁ and Y₂ areselected independently from amino acids having a basic side chain; Z isselected from proline, optionally substituted at γ, β or δ; a natural ornon N-alkylamino acid; a dialkylamino acid; a cyclic dialkylamino acid;pipecolic acid or a derivative thereof; n and i independently are 0 or1; m is an integer between 0 and 3; k is an integer greater than orequal to 3; and Ψ represents a modified peptide bond which issignificantly more resistant to at least one protease than a standardpeptide bond, for preparing a medication intended for the treatment of adiseases involving deregulation of cell proliferation and/orangiogenesis, and preferably acting as a surface nucleolin ligand.

Nucleolin (see structure in FIG. 1A) was initially described as anuclear protein present in the majority of eukaryotic cells. Morerecently, it has been shown that in spite of the absence of atransmembrane domain allowing its attachment to the plasma membrane,another molecular form of this protein is also present on the cellsurface (1-4). This surface nucleolin is closely associated withintracellular actin microfilaments. This association most probably takesplace indirectly via a transmembrane partner.

In the resting cell, nucleolin is found mainly in the nucleolus but alsopartially in the cytoplasm and on the cell surface. Following activationof cell proliferation, cytoplasmic nucleolin is translocated towards themembrane surface by means of an active transport, non-conventionalmechanism independent of the endoplasmic reticulum and Golgi apparatus(1).

The degree of surface nucleolin expression is therefore greatlyincreased following activation of cells, especially activation of cellproliferation. Surface nucleolin therefore constitutes a marker foractivated cells in the proliferation phase. In the particular case ofhuman immunodeficiency virus (HIV), which targets activated cells, ithas been shown that surface nucleolin might be involved in cellinfection by HIV (2,5).

Moreover, it has also been shown that surface nucleolin is expressed atthe surface of tumour cells, such as tumour cells derived from hepaticcarcinoma (6), T-lymphocyte leukaemia (7 and 8) and uterine cancer cells(7), as well as at the surface of activated endothelial cells (9), cellswhich are involved in the angiogenesis process. Moreover, surfacenucleolin constitutes a receptor with weak affinity for various ligands,namely for several growth factors such as midkine (MK), heparin affinregulatory peptide (HARP, also known as pleiotrophin: PTN) andlactoferrin (10-12).

Recently, it was suggested in patent application WO 2005/035579 that itwas possible to treat cancer using nucleolin binding agents, such asanti-nucleolin antibodies, anti-nucleolin interfering RNA or antisenseanti-nucleolin oligonucleotides. The main results presented show thatsurface nucleolins can be considered to be a marker for cancer cells,which in itself does not make for a good target. Only preliminaryresults in mice show that the addition of anti-nucleolin antibodies canimprove tumour regression by taxol. However, no results have beenpublished proving the efficacy of such antibodies alone and the doserequired to obtain this improvement in combination with taxol is notgiven. Moreover, use of antibodies in vivo in humans, as mentionedpreviously, poses serious problems in terms of administration.

Anti-nucleolin agents can act along different pathways. In particular,such agents may or may not act by binding to the protein nucleolin. Forinterfering RNA type agents or antisense anti-nucleolinoligonucleotides, these agents may possibly act at the level ofintracellular nucleic acid and not at the level of binding to nucleolin.For example, in patent application US 2005/0026860, antisense nucleolinoligonucleotides are described as having a positive effect on tumourregression in vivo in a murine model. Nevertheless, the effect observedin mice is partial, with a smaller tumour developing in spite of this,and no effect on angiogenesis is described or suggested. While theseresults suggest that nucleolin could be considered to be a target in thetreatment of cancer, suggest average efficacy not likely to lead to thepossibility of treatment with an anti-nucleolin agent alone but rathersimply as adjuvant treatment in addition to conventional chemotherapy.Moreover, as mentioned earlier, the use of oligonucleotides in vivoposes serious bioavailability problems.

An anti-nucleolin agent can also bind directly to nucleolin protein.Various nucleolin ligands have been described in the literature: peptideF3 (or tumour-homing peptide) is a peptide corresponding to a 34 aminoacid fragment of protein HMG2N which binds to activated endothelialcells of the vessels of various types of tumour. Recently, it has beenshown that peptide F3 binds to nucleolin expressed in endothelial cellsand is then internalised and transported into the nucleus by means of anactive process. Binding to nucleolin and internalisation are blocked byanti-nucleolin antibodies. It has been reported that peptide F3 binds tothe N-terminal area of nucleolin which contains several amino acid richregions (9). In this application, the inventors show that peptide F3 isincapable of inhibiting cell proliferation of NIH-3T3 cells triggered bygrowth factor HARP (see example 1.1.1). Simply binding to surfacenucleolin and being internalised is therefore not enough to confer thecapacity to inhibit tumour cell proliferation on this peptide;

Multivalent compound HB19 (see FIG. 2A), synthesised and described bythe inventors, is also a surface nucleolin ligand (7, 8, 13, 14) whichinteracts with the RGG domain (see FIG. 1B). It has been shown that thiscompound might make it possible to inhibit the infection of cellsactivated by HIV (13) as well as the binding of other natural ligands tonucleolin (10-12). However, no result has been reported demonstratingits possible ability to reduce and/or inhibit tumour growth orangiogenesis; patent application WO 00/61597 described guanine-richoligonucleotides (GROs) as binding to a protein likely to be nucleolinand inhibiting the proliferation of tumour cells in vitro at dosesgreater than or equal to 15 μM (15). In vivo, only the existence of acertain synergy alongside conventional chemotherapy treatment ismentioned. No effect on angiogenesis is described or suggested.Moreover, it would seem that GROs bind mainly to intracellularnucleolin; in an article published by the team which registered patentWO 00/61597, another mixed oligonucleotide called MIX1, which includes Gand T bases such as GROs in their samesense sequence but also A and Cbases, is described as binding to nucleolin with the same efficacy asGROs but with no effect on cell proliferation, which once again suggeststhat simply binding to nucleolin is not enough to confer the ability toinhibit cell proliferation (15); recently, a team has shown that it ispossible to inhibit angiogenesis induced by VEGF by means of apreparation containing anti-nucleolin polyclonal antibodies (16).However, although the anti-nucleolin polyclonal antibody preparationblocks the formation of tubules by endothelial cells, it does not blockthe proliferation of endothelial cells. In addition, only angiogenesisinduced by VEGF was tested whereas there are many other factors involvedin angiogenesis and no result showing inhibition of cell proliferationis described nor suggested.

It is clear from the description above that not all ligands of nucleolinshow anti-tumour activity and none of the above-cited documents suggeststhat any of these ligands is likely to inhibit both the proliferation oftumour cells in general and angiogenesis triggered by various factors.

Moreover, it is crucial to note that the results presented, whethertaken singly or together, in no way suggest that these ligands mightpossess sufficient activity to be used alone, without being combinedwith conventional anti-cancer treatments (radiotherapy or, moreespecially, conventional chemotherapy such as taxol).

However, the inventors have surprisingly shown that the pentavalentpeptide compound HB19, or other compounds with at least 3 pseudopeptideunits of the same type grafted on a support, make it possible to inhibitthe proliferation of tumour cells in general, whether they aredependently or independently anchored (proliferation that ischaracteristic of transformed cells) or triggered by various growthfactors, as well angiogenesis triggered by various factors. Moreover andmore importantly, the inventors show in a murine model that pentavalentcompound HB19 allows in vivo inhibition of both tumour proliferation andangiogenesis but also that the anti-tumour effect of compound HB19 atthe usual dose for a peptide (5 mg/kg) is greater than that of taxol at10 mg/kg, which is one of the standard molecules used in anti-tumourtreatment.

Therefore while the other previously described ligands of surfacenucleolin appear to have only a partial effect likely to lead toadjuvant type treatment, compound HB19 in vivo in mice shows greaterefficacy than taxol, the standard anti-cancer molecule, suggesting thepossibility of using it alone and not in combination with a conventionalchemotherapy molecule. Notably, the taxol dose administered does lead totumour regression but the regression is not total since a tumour wasfound and weighed once mice were sacrified. To the contrary, withmultivalent ligand HB19 at a dose that is 2 times lower, no tumour wasfound in mice after their death, thus demonstrating full regression.

Multivalent compound HB19 therefore appears to be a highly powerfulanti-cancer agent. This effect is probably linked to its dual ability asdemonstrated by the inventors to inhibit both the proliferation oftumour cells, whether triggered by several distinct growth factors oreven independently of anchorage, and the angiogenesis process triggeredby 2 distinct angiogenic factors.

In addition, no toxic effect was found by the inventors, whether oncells cultured in vitro for several weeks in the presence of compoundHB19 or in vivo in mice treated with compound HB19. Moreover,purification of proteins bound to multivalent compound HB19 after invivo administration makes it possible to obtain over 90% surfacenucleolin, suggesting great specificity of interaction betweenmultivalent compound HB19 and nucleolin. This greatly limits thepossibility of the occurrence of side effects. The inventors have alsoshown that although peptide HB19 can be internalised after binding tosurface nucleolin, it does not reach the nucleus, an important fact toexplain the absence of toxicity for healthy cells.

Compound HB19 and derivatives or analogues thereof are also easilysynthesised, even on an industrial scale, under easily controllablehealth safety conditions.

Finally, its specificity for nucleolin as well as for tumour cells andactivated endothelial cells, its pseudopeptide nature and its highsolubility in aqueous media means that it has very good bioavailabilityin vivo. The specificity of compound HB19 for surface nucleolin does notrequire any coupling with a target molecule. Moreover, the presence of amodified peptide bond (reduced in the case of compound HB19) between thelysine and proline of each KPR unit presented in the case of HB19confers on it good resistance to proteases in vivo and an in vivo halflife of over 24 hours, contrary to conventional peptides whose in vivohalf life does not exceed half an hour. In addition, compound HB19 istotally soluble in aqueous media which makes its administration mucheasier as no particular pharmaceutical form is required for itscirculation and targeting in vivo.

Pentavalent compound HB19 therefore presents all the necessarycharacteristics needed to resolve the various technical problems ofsupplying new anti-cancer compounds:

which are capable of having high anti-tumour efficacy alone as a resultof a dual effect on tumour proliferation and angiogenesis, efficacy thatmakes it possible to envisage a single treatment without being combinedwith a conventional chemotherapy molecule such as taxol;

which do not have specificity for a particular type of cancer but rathera broad spectrum of activity against tumour cells and activatedendothelial cells;

which have very few side effects in vivo as a result of specificity fortumour cells and activated endothelial cells compared to healthy cells;

which have a synthesis process that can be easily adapted to anindustrial scale; and

which have sufficient bioavailability in vivo in order not to requirethe development of particular pharmaceutical forms.

The invention therefore relates to the use of a multivalent syntheticcompound comprising or constituted of a support on which is grafted atleast 3 pseudopeptide units, said compound being of formula (I):

Wherein each X independently represents any amino acids;

Y₁ and Y₂ are independently selected from basic natural chain aminoacids;

Z is selected from: proline, possibly substituted at γ, β or δ byhydroxyl, amine, C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ alkynyl, C₅-C₁₂aryl, C₅-C₁₄ aralkyl, C₅-C₁₂ heteroaryl (advantageously C₅ heteroaryl)groups, these groups being themselves possibly substituted by 1 to 6substituents selected from a halogen atom, NO₂, OH, C₁-C₄ alkyl, NH₂,CN, trihalomethyl, C₁-C₄ akyloxy, C₁-C₄ dialkylamino, guanidino group,thiol group;

N-alkylamino acid, natural or not;

dialkylamino acid;

cyclic dialkylamino acid; or

pipecolic acid or derivatives thereof;

n and i are independently 0 or 1;

m is an integer between 0 and 3; k is an integer greater than or equalto 3; and

Ψ represents a modified peptide bond significantly more resistant to atleast one protease than a standard peptide bond, for the manufacture ofa medication intended for the treatment of disorders involvingderegulation of cell proliferation and/or angiogenesis.

Preferably, such a multivalent synthetic compound acts as a ligand forthe surface nucleolin.

In the context of the invention, the term “support” refers to anypharmaceutically acceptable molecule, in other words without intrinsictoxicity, on which at least 3 pseudopeptide units of formula (I) can begrafted. An acceptable support therefore has to be of sufficient size toallow at least 3 pseudopeptide units of formula (I) to be grafted on it,preferably 3 to 8 pseudopeptide units of formula (I). Such an acceptablesupport should also preferably be large enough to allow at least 3,preferably 3 to 8, pseudopeptide units of formula (I) can come togetherto interact in the RGG domain of one or more nucleolin molecules. Inaddition, the support must not be immunogenic.

Such a support can be selected from a linear peptide or cyclic peptide,a peptoid (N-substituted glycine oligomer) that is linear or cyclic, afoldamer (oligomer or polymer with a strong tendency to adopt a compact,well-defined and predictable conformation in solution), a linear polymeror a spherical dendromer (macromolecule consisting or polymers whichcombine according to a tree like process around a multifunctionalcentral core) a sugar or a nanoparticle. Advantageously, said support isselected from a linear or a cyclic peptide or even a linear or cyclicpeptoid.

The use of a linear peptide (see structure of HB19 in FIG. 2A) allowsthe support to be synthesised easily and the results obtained by theinventors with compound HB19 show that such a support does in effectresolve the technical problems posed by this application. A linearpeptide acting as a support in the invention can advantageously containa proportion of lysine greater than 25%. More precisely, when a linearpeptide is used as a support in the invention, the pseudopeptide unitsare preferably grafted in position □□□ of lysine. When a linear peptideis used as the support in the invention, it therefore preferablyincludes at least as many lysine as the number of pseudopeptide unitswhich are to be grafted on.

For example, a support linear peptide can have a sequence selected fromKKKGPKEKGC (SEQ ID NO:1), KKKKGC (SEQ ID NO:2), KKKKGPKKKKGA (SEQ IDNO:3) or KKKGPKEKAhxCONH₂ (SEQ ID NO:4), wherein Ahx represents hexanoicamino acid and CONH₂ represents the fact that the acid group is replacedby an amide group, AhxCONH₂, representing (2S)-2-aminohexanamide, or alinear sequence consisting of 2-4 units (KAKPG, SEQ ID NO :12), namelysequence AcKAKPGKAKPGKAKPGCONH₂ (SEQ ID NO:13, where Ac represents anacetyl group CH₃—CO—, and CONH₂ means that the acid group COOH ofglycine is replaced by an amide group CONH₂). Advantageously, thesupport linear peptide can be peptide KKKGPKEKAhxCONH₂ (see for exampleHB19 in FIG. 2A, SEQ ID NO:5, which has this linear peptide assupport.), or peptide AcKAKPGKAKPGKAKPGCONH₂ (SEQ ID NO:4, where Acrepresents an acetyl group CH₃—CO— and CONH₂ means that the acid groupCOOH of glycine is replaced by an amide group CONH₂, for example, Nucant7 in FIG. 2F, SEQ ID NO :17, which has this linear peptide as asupport).

Among the linear peptides, some are known to adopt a helicoidalstructure. These linear peptides can also be used as supports in theinvention. Such linear peptide supports from a helicoidal structurecomprised of supports consisting of an integer greater than or equal to3, namely 3 to 8, repetitions of the peptide units of sequenceAib-Lys-Aib-Gly (SEQ ID NO :6) or Lys-Aib-Gly (SEQ ID NO :7)respectively where Aib represents 2-amino-isobutyric acid. As each ofthese units consists of a single lysine residue (Lys), as manyrepetitions of these units are needed as are to be grafted onpseudopeptide units of formula (I).

For example, to obtain a pentavalent compound with 5 pseudopeptide unitsof formula (I), the support can be a linear peptide forming a helicoidalstructure of formulaAib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly(SEQ ID NO :8) orLys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly (SEQ ID NO:9). Advantageously, a linear peptide forming a helicoidal structure offormula derived from SEQ ID NO :8 and 9 is used. This formula isselected fromAc-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-CONH₂(SEQ ID NO :18, where Ac represents an acetyl group CH₃—CO— and CONH₂means that the COOH acid group of glycine is replaced by an amide groupCONH₂, see for example Nucant 2 in FIG. 2C, SEQ ID NO :20, which hasthis peptide as a support) orAc-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-CONH₂(SEQ ID NO :19, where the Ac group represents an acetyl group CH₃—CO—and CONH₂ means that the COOH acid group of glycine is replaced by anamide group CONH₂, see for example Nucant 3 in FIG. 2D, SEQ ID NO :21,which has this peptide as a support).

Alternatively, to obtain a hexavalent compound with 6 pseudopeptideunits of formula (I), the support used can be a linear peptide forming ahelicoidal structure of formulaAc-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-Aib-Lys-Aib-Gly-CONH₂(SEQ ID NO :14, where Ac represents a CH₃—CO— group and CONH₂ means thatthe acid group COOH of glycine is replaced by an amide group CONH₂) orAc-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-CONH₂(SEQ ID NO :15, where Ac represents a CH₃—CO— group and CONH₂ means thatthe acid group COOH of glycine is replaced by an amid group CONH₂, seefor example Nucant 6 in FIG. 2E, SEQ ID NO :17, which has this peptideas a support).

A cyclic peptide or peptoid can also be advantageously used as support.In particular, this allows the flexibility of the structure to berestricted. A support cyclic peptide or peptoid can be mainly beselected from hexa-, octa-, deca- or dodeca-cyclic peptide, preferablyconsisting of amino acid residues in the L (levorotatory) and D(dextrorotatory) configuration in alternation (D,L-cyclopeptide) or achain of N-alkyl Glycine residue (cyclic peptoid). An example of acompound with such a support is a cyclic hexapeptide consisting ofalternate alanine (A) residues of configuration D and lysine residues(K) of configuration L with 3 KPR units with a Ψ (Ψ=(CH₂N—)) bondbetween K and P as shown in FIG. 2B (compound Nucant 01).

Advantageously, the support for a compound of formula (I) according tothe invention is a support selected from a cyclic hexapeptide consistingof alternating alkaline (A) residues of configuration D and Lysine (K)residues of configuration L or a linear peptide of sequence SEQ ID NO:1, SEQ ID NO :2, SEQ ID NO :3, SEQ ID NO :4, SEQ ID NO :8, SEQ ID NO:9, SEQ ID NO :13, SEQ ID NO :14, SEQ ID NO :15, SEQ ID NO :18, or SEQID NO :19.

In the context of the invention, the term “grafted” for thepseudopeptide units means being bound to the support by means of acovalent bond, either directly or through the intermediate of a spacercompound between the pseudopeptide and support. As a result of this, inone particular embodiment, the pseudopeptide units are grafted directlyon the support without a spacer compound between them and the support.In another embodiment, the pseudopeptide units are grafted on thesupport through the intermediate of a spacer. Examples of acceptablespacers include compounds of the type ethylene glycol, piperazine or anamino acid of the type aminohexanoic acid or beta-alanine.

In the case where the support is a linear or cyclic peptide and wherethe pseudopeptide units are grafted directly on the peptide, bondingbetween the peptide and the pseudopeptide units is preferably carriedout at the lysine residue of the peptide support, at the amino group inthe α or ε position, preferably at the amino group in the ε position (onthe side chain) of lysine. Thus, direct grafting of pseudopeptide unitson the peptide support is advantageously carried out by means of anamide bond between the acid group COOH of the amino acid in theC-terminal position of the pseudopeptide unit and an amino group of thelysine residue, preferably the amino group in the e position (on theside chain) of lysine.

In the compounds according to the invention, at least 3 pseudopeptideunits are grafted on the support. In fact, the inventors' results showthe importance of binding to the RGG domain of nucleolin (see FIG. 1)for exceptional anti-tumour efficacy of compound HB19 and derivativecompounds or analogues. Binding to the RGG domain of nucleolin isobtained by means of multivalent presentation of several pseudopeptideunits such as those incorporated into formula (I). For compounds forwhich the support is a linear peptide of sequence KKKGPKEKGC, KKKKGC,KKKKGPKKKKGA or KKKGPKEKAhxCONH₂, the inventors have shown that below 3units (k<3), the efficacy of binding to nucleolin is lower andanti-tumour efficacy is probably less. The compounds according to theinvention therefore include at least 3 pseudopeptide units grafted onthe support, k being an integer greater than or equal to 3. Thecompounds according to the invention therefore advantageously present3-8 pseudopeptide units (3≦k≦8) grafted on the support. Moreover, theinventors have shown that activity was optimal with 5 or 6 pseudopeptideunits grafted on the support (k=5), since the efficacy of binding tonucleolin does not increase with a higher number of pseudopeptide units.Advantageously, in the compounds of formula (I), k is therefore between3 and 8, preferably between 4 and 7, between 4 and 6, between 4 and 5,or between 5 and 6. Even more advantageously, in compounds of formula(I), k is equal to 5 or even better 6.

In the context of the invention, the term “any amino acid” means anynatural or synthetic amino acid, possibly modified by the presence ofone or more substituents. More precisely the term amino acid means analpha aminated amino acid with the following general structure:

where R represents the side chain of the amino acid. In the context ofthe invention, R therefore represents the side chain of a side ornon-side amino acid. The term “natural amino acid” means any amino acidwhich is found naturally in vivo in a living being. Natural amino acidstherefore include amino acids coded by mRNA incorporated into proteinsduring translation but also other amino acids found naturally in vivowhich are a product or by-product of a metabolic process, such as forexample ornithine which is generated by the urea production process byarginase from L-arginine. In the invention, the amino acids used cantherefore be natural or not. Namely, natural amino acids generally havethe L configuration but also, according to the invention, an amino acidcan have the L or D configuration. Moreover, R is of course not limitedto the side chains of natural amino acid but can be freely chosen.

In the pseudopeptide units of compounds of formula (I), Z is eitherabsent (i=0), or present (i=1) and is then selected from:

proline, possibly substituted at γ, β or δ by hydroxyl groups, amine,C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ alkynyl, C₅-C₁₂ aryl, C₅-C₁₄aralkyl, C₅-C₁₂ heteroaryl (advantageously a C₅ heteroaryl), thesegroups themselves possibly being substituted by 1 to 6 substituentsselected from a halogen atom, NO₂, OH, C₁-C₄ alkyl, NH₂, CN,trihalomethyl, C₁-C₄ akyloxy, C₁-C₄ dialkylamino, guanadino group, thiolgroup; N-alkylamino acid, natural or not; dialkylamino acid (for exampleisobutyric amino acid); cyclic dialkylamino acid; or pipecolic acid orderivatives thereof.

The term “C₁-C_(i) alkyl” means a linear or branched saturatedhydrocarbon radical of formula —C_(j)H_(2j+1), where 1≦j≦i. The C₁-C₁₀alkyl therefore includes C₁ alkyls (methyl), C₂ (ethyl), C₃ (n-propyl,or isopropyl), C₄ (n-butyl, isobutyl, sec-butyl or tert-butyl), C₅ (eg:n-pentyl, neopentyl, isopentyl, tert-pentyl), and C₆ to C₁₀ alkyls. Theterm “C₁-C₁₀ alkanyl” means a linear or branched unsaturated hydrocarbonradical consisting of 1 to 10 carbon atoms and including at least oneC═C double bond. The term “C₁-C₁₀ alkynyl” means a linear or branchedunsaturated hydrocarbon radical with 1 to 10 carbon atoms and at leastone C≡C triple bond. The term “C₅-C₁₂ aryl” means an aromatic polycyclicor monocyclic hydrocarbon radical with 5-12 carbon atoms. The term“C₅-C₁₄ alalkyl” means a combination of an alkyl and an aryl with atotal of 5 to 14 carbon atoms. The term “C₅-C₁₂ heteroaryl” means anaryl group where at least one carbon atom on the hydrocarbon chainnormally carrying 5 to 12 carbon atoms is substituted by another atomselected from N, O, or S. The term “C₅ heteroaryl” therefore means anaryl group where at least 1 of the 5 carbon atoms on the hydrocarbonchain is substituted by another atom selected from N, O or S. The term“C₁-C₄ akyloxy” means a group of formula —O(O)C—(C₁-C₄ alkyl),—O(O)C—(C₄-C₁₂cycloalkyl), —O(O)C—(C₄-C₁₂ aryl), —O(O)C—(C₄-C₁₂arylalkyl, or —O(O)C—(C₄-C₁₂ heteroaryl). Advantageously, in thecompound of formula (I), such an “C₁-C₄ akyloxy” is selected from thegroup of formula —O(O)C—(C₁-C₄ alkyl), —O(O)C—(C₄ cycloalkyl),—O(O)C—(C₄ aryl) —O(O)C—(C₄ arylalkyl), or —O(O)C—(C₄ heteroaryl). Theterm “C₁-C₄ dialkylamino” means a radical of formula —N(C₁-C₄ alkyl)₂where each alkyl is identical or different.

The term “N-alkylamino acid” means any amino acid in which one of thehydrogen atoms in the amine group is substituted by a C₁-C₁₀ alkyl chainor a C₅-C₁₄ arylalkyl group, preferably C₅-C₁₀, namely C₁₀, possiblysubstituted. Examples of N-alkylamino acids include N-methylglycine orsarcosine, N-methylisoleucine acid, N-methylvaline acid, etc . . . Theterm “dialkylamino acid” means any amino acid in which 2 hydrogen atoms(on the central carbon or amine groups) are substituted by a C₁-C₁₀alkyl chain or a C₅-C₁₄ arylalkyl group, preferably C₅-C₁₀, namely C₁₀,possibly substituted. Examples of dialkylamino acids include2-amino-isobutyric acid (Aib), aminocyclopropane carboxylic acid, etc.

Advantageously, Z is present and therefore i=1. Also advantageously,when Z is present (i=1), then Z is a proline, possibly substituted at γ,β or δ as described previously.

In the pseudopeptide units of the compound of formula (I), Y₁ and Y₂ areselected from amino acids with a basic side chain. The term “amino acidwith a basic side chain” means any natural or nonnatural amino acidwhose side chain R has a pKa value greater than 7 (pKa(R)>7). Thus, anyamino acid can be used for Y₁ and Y₂, as long as its side chain has apKa value greater than 7, preferably greater than 7.5, greater than 8,greater than 8.5 or greater than 9. In particular, among the naturalamino acids those whose side chain has a pKa value greater than 7include lysine (K, pKa(R)≈10.5), arginine (R, pKa(R)≈12.5), ornithine(inferior homologue of lysine, pKa(R)≈10.8), generally considered to benatural basic amino acids. Thus, in an advantageous embodiment, Y₁ andY₂ are independently selected from arginine (R), lysine (K) andornithine. Even more advantageously, Y₁ is a lysine (K) and Y₂ is anarginine (R). However, other non-natural amino acids can be used insteadas long as the pKa value of their side chain R is greater than 7,preferably greater than 7.5, greater than 8, greater than 8.5, orgreater than 9.

In the compounds of the invention, the pseudopeptide unit that isessential for binding to the RGG domain of nucleolin is the sub-unit offormula (II)

wherein Y₁ and Y₂ are as defined above. Nevertheless, the presence atone or the other end of this essential sub-unit consisting of severalamino acids as defined above is not such that it would prevent bindingto nucleolin. This is why the essential sub-unit of formula (II) caninclude at one and/or the other end 0 to 3 of any amino acidsrepresented in the formula (I) by (X)n and (X)m respectively, where n isequal to 0 or 1 and m is an integer between 0 and 3. Advantageously, thenumber of the amino acids present at one and/or other end of theessential sub-unit of formula (II) is low, in other words, n isadvantageously 0 and m is advantageously an integer between 0 and 2,advantageously 0 or 1, advantageously 0. Thus in an advantageousembodiment, n and m are equal to 0.

In the compounds of the invention, the sub-unit of formula (II) includesa modified peptide bond Ψ, significantly more resistant to at least oneprotease than a standard peptide.

The term “standard peptide bond” means an amide bond of formula (—CONH—)which is normally present between 2 amino acids in a natural protein.Such a bond is sensitive to the action of proteases. The term “modifiedpeptide bonds Ψ” means a chemical bond between 2 amino acids of chemicalformula distinct from the standard peptide bond of formula (—CONH—).This modified bond Ψ is such that it is significantly more resistant toat least one protease than a standard peptide bond of formula (—CONH—).The term “protease”, also known as “peptidase” or “proteolytic enzyme”,means any enzyme which cleaves the standard peptide bonds in proteins.This process is known as proteolytic cleavage. This involves the use ofa water molecule which is what leads to proteases being classified ashydrolases. The proteases namely include proteases known as N-peptidaseswhich carry out the cleavage of the N-terminal end of proteins. Theseproteases are particularly inconvenient in terms of the in vivostability of peptides without modified peptide bonds. This is whypseudopeptide units of the compounds of formula (I) include a modifiedbond Ψ between Y₁ and Z (if i=1) or Y₁ and Y₂ (if i=0) such that theresistance of the sub-unit of formula (II) is significantly increasedwhich is essential for binding to nucleolin, namely to theseN-peptidases. The Ψ bond should therefore make it possible tosignificantly increase resistance to at least one N-peptidase. Thismakes it possible to significantly increase the half-life of compoundsof formula (I) in vivo and in vitro. Namely, compound HB19 which has amodified bond Ψ, has a half-life of more than 24 hours in human serum orfoetal calf serum at 37° C. whereas the same compound with a standardpeptide bond instead of the Ψ bond only has a half-life of one hourunder these same conditions.

Moreover, the inventors have found that the presence of this modifiedbond Ψ also makes it possible to significantly increase the efficacy ofbinding to nucleolin. This phenomenon may be due to the fact that thisallows compound HB19 to form an irreversible complex with nucleolin.

Various chemical bonds likely to significantly increase resistance to atleast one protease are known. Thus, in an advantageous embodiment, Ψrepresents a reduced bond (—CH₂NH—) or (—CH₂N—) in the case wherebonding takes place at the level of a secondary amine group as is thecase with the bond with proline), a retro-inverso bond (—NHCO—), amethyleneoxy bond (—CH₂—O—), a thiomethylene bond (—CH₂—S—), a carbabond (—CH₂—CH₂—), a ketomethylene bond (—CO—CH₂—), a hydroxyethylenebond (—CHOH—CH₂—), a (—N—N—) bond, an E-alkene bond or a (—CH═CH—) bond.Namely, the inventors have shown that using a reduced bond (—CH₂—NH—)makes it possible to significantly increase resistance to at least oneprotease. Advantageously, Ψ therefore represents a reduced bond(—CH₂NH—).

Although only the Ψ between Y₁ and Z (if i=1) or Y₁ and Y₂ (if i=0) issystematically present in compounds of formula (I), it is also possiblethat other peptide bonds of the pseudopeptide units may be modified asdescribed earlier. In particular, in the context of the invention, thebonds between the amino acids which are not specified can equally wellbe standard peptide bonds or modified Ψ bonds as described earlier. Thepresence of additional Ψ bonds may make it possible to further increaseresistance to proteases of compounds of formula (I). Nevertheless, theincrease linked to the presence of the first Y bond between Y₁ and Z (ifi=1) or Y₁ and Y₂ (if i=0) is already highly significant and theaddition of other Ψ bonds complicates synthesis of the pseudopeptideunits and therefore of compounds of formula (I). The presence ofadditional Ψ bonds is therefore possible but optional.

Examples of compounds that can be used in the invention include inparticular the compounds (see FIG. 2 and examples 1, 2, 3 and 4):

HB19 (FIG. 2A, SEQ ID NO : 5, a compound which has as a support a linearpeptide of SEQ ID NO :4 in which the 5 pseudopeptide units K Ψ PR (withΨ=CH₂—N) are covalently bound to the ε amino group of each of the 5lysine residues),

Nucant 01 (FIG. 2B), a compound which has a support a cyclic hexapeptideconsisting of alternating alanine residues (A) of configuration D andlysine residue (K) of configuration L, where the 3 pseudopeptide units KΨ PR (with Ψ=CH₂—N) are covalently bound to the ε amino group of each ofthe 3 lysine residues (K); see FIG. 2B),

Nucant 2 (FIG. 2C, SEQ ID NO : 20, a compound which has as a support alinear peptide with a helicoidal structure of sequence SEQ ID NO :18 inwhich 5 pseudopeptide units K Ψ PR (with Ψ=CH₂—N) are covalently boundto the ε amino group of each of the 5 lysine residues),

Nucant 3 (FIG. 2D, SEQ ID NO : 21, a compound which has as a support alinear peptide with a helicoidal structure of sequence SEQ ID NO :19 inwhich 5 pseudopeptides K Ψ PR (with Ψ=CH₂—N) are covalently bound to theε amino group of each of the 5 lysine residues),

Nucant 6 (FIG. 2E, SEQ ID NO : 16, a compound which has as a support alinear peptide with a helicoidal structure of sequence SEQ ID NO :15 inwhich 6 pseudopeptide units K Ψ PR (with Ψ=CH₂—N) are covalently boundto the ε amino group of each of the 6 lysine residues),

Nucant 7 (FIG. 2F, SEQ ID NO : 17, a compound which has a support alinear peptide of sequence SEQ ID NO :13 in which 6 pseudopeptide unitsK Ψ PR (with Ψ=CH₂—N) are covalently bound to the ε amino group of eachof the 6 lysine residues).

The compounds described above are used for the manufacture of amedication for use in the treatment of a disease involving deregulationof cell proliferation and/or angiogenesis. The term “disease involvingderegulation of cell proliferation and/or angiogenesis” means, in thecontext of the invention, any human or animal disease affecting one ormore organs in which one or more abnormal cell proliferation phenomenaare observed, as well as groups of cells or tissues and/or abnormalneovascularisation. Evidently, such diseases include all types ofcancer, such as adenoma, sarcoma, carcinoma, lymphoma, and especiallycancer of the ovary, breast, pancreas, lymphatic ganglion, skin, blood,lung, brain, kidney, liver, nasopharyngeal cavity, thyroid, centralnervous system, prostate, colon, rectum, uterine neck, testicles orbladder. They also include noncancerous diseases of the skin such asepidermal or dermal cysts, psoriasis, angiomas, as well as oculardiseases such as age related macular degeneration (ARMD), diabeticretinopathy or neovascular glaucoma. Neurodegenerative diseases such asmultiple sclerosis, Parkinson's and Alzheimer's or autoimmune diseasessuch as lupus or rheumatoid polyarthritis, as well as diseases relatedto atherosclerosis.

Advantageously, said disease involving deregulation of cellproliferation and/or angiogenesis is cancer, in particular one of thosecited above.

The invention also relates to a method for screening molecules thatinhibit both cell proliferation and angiogenesis, comprising:

contacting cells expressing surface nucleolin with a test molecule, and

determining the capacity of said test molecule to bind to the RGG domainof nucleolin.

It is possible to produce a synthetic RGG domain of 60 amino acids ofnucleolin by chemical synthesis or by using genetic engineering via theexpression of its nucleic DNA sequence. Determination of the capacity ofsaid test molecule to bind to the RGG domain of nucleolin can then becarried out by various technologies known to the man skilled in the art.

Notably, they can be carried out by measuring binding to a synthetic RGGdomain of 60 amino acids using a surface plasmonic resonance technique,in particular with a Biacore® 3000 apparatus. The BIACORE® system is abiosensor using the physical principle of surface plasmonic resonance(SPR). It allows measurement in real time and without specific labellingof the kinetic constants of interaction (Ka and Kd) between twomolecules on a biospecific surface. To this end, one of the molecules(ligand) is immobilized on the sensor surface and the other (analyte) isinjected. The principle of detection by SPR is quantification of changesin the refractive index close to the surface, linked to variations inthe mass on the surface of the biosensor resulting from the formationand dissociation of molecular complexes. When monochromatic, polarizedlight arrives at the interface between two media with differentrefractive indexes and this interface is coated with a fine layer ofmetal, the intensity of reflected light is clearly reduced for aparticular incidence angle. This results from the fact that oneelectromagnetic component of light, the evanescent wave, is propagatedperpendicularly to the interface, up to 1 μm. The resonance anglesvaries, namely as a function of the weight of molecules located near thesurface. Consequently, monitoring of the SPR angle as a function of timemakes it possible to observe association and dissociation of the ligandand analyte in real time. The signal obtained is recorded (sensogram).It is quantified in resonance units (RU). A change of 1000 RUcorresponds to a 0.1° shift in the angle and is equivalent to binding of1 ng of protein per mm². This technology therefore makes it possible notonly to establish the capacity of the test molecule to bind to the RGGdomain but also the efficacy of binding (affinity constant) of saidmolecule to the RGG domain of nucleolin.

It is also possible to generate a synthetic RGG domain labelled withbiotin or fused to a peptide or protein such as GST (GlutathioneS-transferase). The presence of biotin or GST then makes it possible todetect, by means of a labelled avidine/streptavidine ligand(fluorescent, luminescent, radioactive, etc.), whether the RGG domain isbiotinylated and/or if there are anti-GST antibodies if the RGG domainis fused with GST. Although less accurate, these technologies allowfaster and easier screening of a larger number of molecules for theircapacity to bind to the RGG domain of nucleolin, after which moreprecise determination of the efficacy of binding to the RGG domain ofnucleolin can be carried out using the previously mentioned surfaceplasmonic resonance technique.

Thus, it is preferable to firstly carry out quick and easy screening ofthe capacity to bind to RGG domain of nucleolin. Next, the analysis isrefined by determining the efficacy of binding to the RGG domain ofcandidates capable of binding to the RGG domain by the surface plasmonicresonance technique. However, in the case where only a small number ofcompounds are to be tested, their efficacy of binding to the RGG domaincan be measured directly using the surface plasmonic resonancetechnique.

The invention further relates to a compound as defined earlier, with theexception of compounds whose support is a non-cyclic peptide includingan amino acid sequence selected from KPG, KGP, KGC, or KX₁KX₄KX₁K, whereX₁ is optional and selected from lysine (K), valine (V), alanine (A),glutamic acid (E) and isoleucine (I), and X₄ is optional and selectedfrom valine (V), alanine (A), glutamic acid (E) and isoleucine (I).

Notably, the support (with the exception of those excluded in thepreceding paragraph), the manner in which the pseudopeptide units aregrafted on the support and amino acids X, Y₁ and Y₂, n and m, or Y canbe in the form of any previously-described embodiment.

The invention also relates to a compound as described earlier with theexception of compounds whose support is a linear peptide. In particular,the support (with the exception of linear peptides), the manner in whichthe pseudopeptide units are grafted on the support and amino acids X, Y₁and Y₂, n and m, or Y can be in the form of any previously-describedembodiment.

Notably, the support can be selected from a linear peptide comprising anamino acid sequence selected from Aib-K-Aib-G (SEQ ID NO : 6) or K-Aib-G(SEQ ID NO : 7), a cyclic peptide, a linear or cyclic peptoid, afoldamer, a linear polymer or spherical dendrimer, a sugar or ananoparticle.

In some advantageous cases, the support can be a linear peptideconsisting of an amino acid sequence selected from SEQ ID NO : 8 and SEQID NO : 9, SEQ ID NO : 14, SEQ ID NO : 15, SEQ ID NO :18, and SEQ ID NO:19.

The compounds according to the invention can include, in particular,compounds whose support is a cyclic peptide such as Nucant 01 (FIG. 2B),or a linear peptide with a helicoidal structure such as Nucant 2 (SEQ IDNO : 20 and FIG. 2C) and Nucant 3 (SEQ ID NO : 21 and FIG. 2D),described earlier. They can also include the compound Nucant 6 (SEQ IDNO : 16, see example 4 and FIG. E).

The invention also relates to the compound Nucant 7 (SEQ ID NO : 17 andFIG. 2F) as the compound.

The invention further relates to a compound as defined earlier, with theexception of compounds whose support is a non-cyclic peptide includingan amino acid sequence selected from KPG, KGP, KGC, or KX₁KX₄KX₁K, whereX₁ is optional and selected from lysine (K), valine (V), alanine (A),glutamic acid (E) and isoleucine (I), and X₄ is optional and selectedfrom valine (V), alanine (A), glutamic acid (E) and isoleucine (I).

Notably, the support (with the exception of those excluded in thepreceding paragraph), the manner in which the pseudopeptide units aregrafted on the support and amino acids X, Y₁ and Y₂, n and m, or Y canbe in the form of any previously-described embodiment.

The invention also relates to a compound as described earlier with theexception of compounds whose support is a linear peptide for use asmedication. In particular, the support (with the exception of linearpeptides), the manner in which the pseudopeptide units are grafted onthe support and amino acids X, Y₁ and Y₂, n and m, or Y can be in theform of any previously-described embodiment.

Notably, the support can be selected from a linear peptide comprised ofan amino acid sequence selected from Aib-K-Aib-G (SEQ ID NO: 6) orK-Aib-G (SEQ ID NO: 7), a cyclic peptide, linear or cyclic peptoid,foldamer, linear polymer or spherical dendrimer, sugar or nanoparticle.

In some advantageous cases, the support can be a linear peptideconsisting of an amino acid sequence selected from SEQ ID NO: 8 and SEQID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:18, and SEQ ID NO: 19.

Such compounds, for use as medication, particularly include compoundswhose support is a cyclic peptide such as Nucant 01 (FIG. 2B), or alinear peptide with a helicoidal structure such as Nucant 2 (SEQ ID NO:20 et FIG. 2C) and Nucant 3 (SEQ ID NO: 21 and FIG. 2D), describedearlier. They can also include the compound Nucant 6 (SEQ ID NO: 16, seeexample 4 and FIG. 2E).

The invention also relates to the compound Nucant 7 (SEQ ID NO: 17 andFIG. 2F) for use as medication.

The invention further relates to a pharmaceutical composition comprisinga compound as defined earlier, with the exception of compounds whosesupport is a non-cyclic peptide including an amino acid sequenceselected from KPG, KGP, KGC, or KX₁KX₄KX₁K, where X₁ is optional andselected from lysine (K), valine (V), alanine (A), glutamic acid (E) andisoleucine (I), and X₄ is optional and selected from valine (V), alanine(A), glutamic acid (E) and isoleucine (I).

Notably, the support (with the exception of those excluded in thepreceding paragraph), the manner in which the pseudopeptide units aregrafted on the support and amino acids X, Y₁ and Y₂, n and m, or Y canbe in the form of any previously-described embodiment.

The invention also relates to a pharmaceutical composition as describedearlier with the exception of compounds whose support is a linearpeptide. In particular, the support (with the exception of linearpeptides), the manner in which the pseudopeptide units are grafted onthe support and amino acids X, Y₁ and Y₂, n and m, or Y can be in theform of any previously-described embodiment.

Notably, the support can be selected from a linear peptide comprised ofan amino acid sequence selected from Aib-K-Aib-G (SEQ ID NO : 6) orK-Aib-G (SEQ ID NO : 7), a cyclic peptide, linear or cyclic peptoid,foldamer, linear polymer or spherical dendrimer, sugar or nanoparticle.

In some advantageous cases, the support can be a linear peptideconsisting of an amino acid sequence selected from SEQ ID NO: 8 and SEQID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:18, and SEQ ID NO:19.

The pharmaceutical compositions according to the invention can include,in particular, compounds whose support is a cyclic peptide such asNucant 01 (FIG. 2B), or a linear peptide with a helicoidal structuresuch as Nucant 2 (SEQ ID NO: 20 et FIG. 2C) and Nucant 3 (SEQ ID NO: 21and FIG. 2D), described earlier. They can also include the compoundNucant 6 (SEQ ID NO: 16, see example 4 and FIG. 2E).

The invention also relates to a pharmaceutical composition containingthe compound Nucant 7 (SEQ ID NO: 17 and FIG. 2F).

Such pharmaceutical compositions combine the compound(s) according tothe invention with a pharmaceutically acceptable support.

This invention also relates to the use of a synthetic multivalentcompound including or consisting of a support on which at least 3pseudopeptide units are grafted, said compound being of formula (I):

wherein each X independently represents any amino acid; Y₁ and Y₂ areindependently selected from basic side chain amino acids; Z is selectedfrom proline, possibly substituted at γ, β or δ; a natural or nonN-alkylamino acid; dialkylamino acid; cyclic dialkylamino acid;pipecolic acid or derivatives thereof; n and i are independently 0 or 1;m is an integer between 0 and 3; k is an integer greater than or equalto 3 and Ψ represents a modified peptide bond, significantly moreresistant to at least one protease than a standard peptide bond, for themanufacture of a medication intended for the treatment of inflammatorydiseases.

It is now well-established that chronic inflammatory diseases, namelyautoimmune diseases with cellular mediation, are partly triggered bycytokines. Results obtained with various experimental animal models haverevealed the role played by cytokines in the pathogenesis of disease(17). For example, proinflammatory cytokines such as tumour necrosisfactor α (TNF-α), interleukin 1 (IL-1), IL-6, IL-15 and IL-18 regulatethe immune and inflammatory responses in patients with rheumatoidpolyarthritis. In particular, TNF-α and IL-1β promote the destruction ofcartilage and bone marrow.

Moreover, in the course of the inflammatory process, the vascularendothelium expresses different chemokines and adhesion molecules whichparticipate in the recruitment of leukocytes in the inflammatory focus(18).

IL-8 is a C—X—C chemokine which triggers the activation and selectiverecruitment of leukocytes in tissues which are the site of inflammation.When high levels are expressed, IL-8 can have pathological consequencesfor the body. Lipopolysaccharide (LPS) and proinflammatory cytokinessuch as TNF-α and IL-1 trigger the secretion of IL-8 by many cell types,particularly endothelial cells (19).

Intercellular Adhesion Molecule-1 (ICAM-1) is an immunoglobulin typeprotein expressed at the surface of several cell types includingendothelial cells and cells involved in the immune response. It plays animportant role in the adhesion and migration of leukocytes to the sitesof inflammation (20).

Proinflammatory cytokines are also involved in the general inflammatoryresponse (septic shock) triggered by bacterial infections.Lipopolysaccharide (LPS) is an integral component of the externalmembrane of gram-negative bacteria. This immuno-stimulating molecule isa factor which largely contributes to triggering the generalinflammatory response, called septic shock, which often accompaniesgram-negative bacterial infections. LPS has the biological property ofstimulating the production of cytokines, such as TNF-α, IL-1 and IL-6,by the lymphoreticular cells. The induction of these cytokines plays apivotal role in the development of septic syndrome because theadministration of TNF-α alone can lead to a septic condition and deathsince TNF-α can trigger the production of IL-1 and IL-6 in vivo.Moreover, in animal models, pretreatment with anti-TNF-α antibodies andthe IL-1 receptor agonist makes it possible to protect animals againstthe lethal effects of LPS (21).

Severe septicaemia is associated with an aggressive inflammatoryreaction and organ deficiencies. It is frequently linked to a highmortality rate. It can follow on from a bacterial, fungal or viralinfection. This reaction is marked by sequential secretion ofproinflammatory then inflammatory cytokines. TNF-α and IL-β are amongthe most proinflammatory cytokines. Up until now, no clinical trialsinvolving anti-LPS or anti-cytokine reagents have met with success (22,23).

The proinflammatory cytokines also appear to play a physiopathologicalrole in patients with carditis or inflammation of the heart, whichmanifests itself in the form of inflammation of the endocardium(endocarditis) or pericardium (pericarditis) or cardiac muscle(myocarditis). For example, serum levels of IL-6 are significantlyhigher in patients with infectious endocarditis which can be caused, inparticular, by a Staphylococcus aureus infection. In this way, high IL-6levels in the serum can suggest the existence of infectious pericarditisand can be used as a tool for the diagnosis and follow-up of treatmentfor this disease (24, 25)

In view of the important of the role of proinflammatory cytokines suchas TNF-α, IL-1 and IL-6 in inflammatory disease, anti-inflammatorycytokine therapies which involve anti-TNF-α, anti-IL-1 and anti-IL-6reagents have been developed for the treatment of patients sufferingfrom inflammatory diseases (26).

Various clinical trials involving anti-inflammatory cytokine reagents inthe treatment of chronic inflammatory diseases, such as rheumatoidarthritis and abdominal inflammatory disease, have met with somesuccess: Etanercept (TNF receptor-P75 Fc fusion protein), Infliximab(chimeric human anti-TNF-α monoclonal antibody), Adalimumab (recombinanthuman anti-TNF-α monoclonal antibody) and Anakinra (recombinant form ofhuman IL-1β receptor antagonist) (22).

Nevertheless, all the anti-inflammatory cytokine reagents available todate are proteins and thus present the disadvantages associated withprotein medications, in particular, the high cost of production andproblems linked to largescale production.

Consequently, there is a real need for low molecular weight moleculescapable of specifically targeting the synthesis pathways ofproinflammatory cytokines.

Surprisingly, the inventors have found that compounds of formula (I),such as those described earlier, have anti-inflammatory activity; inparticular, they inhibit the production of TNF-α, IL-6 and IL-8 as wellas the expression of ICAM-1 by various cell types stimulated by LPS.These compounds are extremely interesting because, as mentioned earlier:

no toxic effect of these compounds has been observed by the inventor,neither in vitro nor in vivo;

these compounds are easy to synthesize, even on an industrial scale,under easily controlled conditions;

these compounds have sufficient in vivo bioavailability of themselvesnot to need a particular pharmaceutical form to be developed.

The invention thus relates to the use of a synthetic multivalentcompound of formula (I), as defined earlier in any of the embodimentsdescribed above, for the preparation of a medication intended for thetreatment of inflammatory diseases.

In particular, in formula (I), the support, the number of pseudopeptideunits k, the manner in which the pseudopeptide units are grafted on thesupport, the amino acids X, Y1 and Y2; n and m, or Ψ can be in the formof any embodiment described above.

The term <<inflammatory disease>> means any disease in which aninflammatory reaction has pathological consequences for the organism. Inparticular, inflammatory diseases in the context of the inventioninclude autoimmune diseases (such as lupus or rheumatoid polyarthritis),septicaemia, septic shock, cardiac inflammatory diseases (carditis, andespecially endocarditis, pericarditis, myocarditis, in particular thoseof an infectious origin such as those caused by Staphylococcus aureus),graft rejection, trauma, inflammatory diseases of the joints (notably,different forms of arthritis), inflammatory diseases of thegastrointestinal system (notably, colitis, enteritis, gastritis,gastroenteritis, and chronic inflammatory diseases of the intestine suchas Crohn's disease and haemorrhagic rectocolitis (HRC)), inflammatorydiseases of the skin (eczema, allergic contact dermatitis, psoriasis,dermatosis), inflammatory diseases of the respiratory system, especiallychronic obstructive pulmonary disease (COPD), and allergies.

In an advantageous embodiment, the inflammatory disease is an autoimmunedisease, in particular lupus or rheumatoid arthritis. In anotheradvantageous embodiment, the inflammatory disease is septic shock. Inyet another advantageous embodiment, the inflammatory disease is anendocarditis, particularly endocarditis of infectious origin, such asthat caused by Staphylococcus aureus.

The advantages of this invention are illustrated in the figures andexamples given below.

EXAMPLES

Anti-Tumour Activity of Pentavalent Compound HB19

Effect of Pentavalent Compound HB19 on the Growth of Tumour Cells inVitro

Role of Surface Nucleolin in Anchorage-Dependent Cell Proliferation andInhibitory Effect of HB-19 on this Proliferation

The role played by nucleolin in the biological activity of the HARPmolecule was studied in a series of experiments: the mitogenic activityof HARP, tested by measuring the incorporation of tritiated thymidine byNIH 3T3 cells, was evaluated in the presence or absence of a monoclonalantibody which specifically recognizes nucleolin. The results show thatthis antibody inhibits the mitogenic activity of HARP in NIH 3T3 cellsin a dose-dependent manner (FIG. 3A).

The addition of 50 nM of anti-nucleolin antibody completely inhibitsmitogenic activity resulting from 4 nM of HARP whereas non-specificantibodies against nucleolin of the same isotype have no effect onproliferation induced by HARP. This is the case whatever theimmunoglobulin concentration used, thus demonstrating the specificity ofthe inhibition observed (FIG. 3B).

Peptide F3 and compound HB19 are two ligands of nucleolin. Peptide F3binds to the N-terminal part of nucleolin which contains many acid aminoacid regions (9), contrary to compound HB19 which binds to the RGGdomain located in the C-terminal part of nucleolin (see FIG. 1). It hasalso been shown that specific binding of compound HB19 to the surface ofcells is not affected by the presence of peptide F3 (experimentconducted by the inventors using FACS).

The inventors investigated whether peptide F3 and pentavalent compoundHB19 were capable of inhibiting cell proliferation of NIH-3T3 cellstriggered by the growth factor HARP. In the same series of experiments,the effects of compound HB19 and peptide F3, which specifically bind tosurface nucleolin, were therefore tested.

The results for peptide F3 are given in FIG. 3C and unequivocally showthat peptide F3, like IgG antibodies, does not lead to inhibition of NIH3T3 proliferation triggered by HARP.

The results for HB19 are given in FIG. 3D and show that HB-19 leads todose-dependent inhibition of NIH 3T3 proliferation triggered by HARP.Addition of 0.5 μM of HB-19 leads to 81% inhibition of the effecttriggered by 4 nM of HARP. This clearly shows that it is not enough tohave a nucleolin ligand capable of being internalised to bring aboutinhibition of proliferation and suggests that, in order to be effective,a nucleolin ligand needs to be multivalent and bind to one or more RGGunits in the C-terminal domain of one or more nucleolin molecules.

Moreover, it is interesting to note that comparable inhibition isobserved when cells are pretreated for one hour with varyingconcentration of HB-19, washed then stimulated by HARP (FIG. 4). Thisresult shows that HB-19 binds to surface nucleolin present on NIH 3T3,thus blocking cell proliferation induced by HARP one hour later.

In order to study whether the effect of HB-19 on the inhibition of cellproliferation is specific for a given growth factor such as HARP, twoseries of experiments were carried out which consisted in studying theeffect of HB-19 on NIH 3T3 cells stimulated by:

FGF-2, another growth factor, or

5% foetal calf serum containing a mixture of various growth factors.

The results of these experiments are presented in FIG. 5 and show thatHB-19 at a concentration de 0.5 μM is capable of inhibiting theproliferation of cells stimulated by FGF-2 (A) or by foetal calf serum(B).

Thus, the results show that overall, HB-19 is capable of in vitroinhibition of the proliferation of tumour cells. This is the casewhatever the agent used to trigger cell proliferation.

Role of Nucleolin in Anchorage-Dependent Cell Proliferation andInhibitory Effect of HB-19 on this Proliferation

In parallel to these studies on cell proliferation, the role ofnucleolin on anchorage-independent growth, the phenotype characteristicof transformed cells, was tested in a growth model on wet agar using thehuman mammary carcinoma line, MDA-MB-231, as well as a mouse melanomaline, B16-BL6.

In these experiments, cells were cultured on agar gel in the presence orabsence of varying concentrations of the control anti-nucleolinantibodies, control immunoglobulins or compound HB-19. After 10 days ofincubation at 37° C., the number of colonies present in each cultureplate was counted. As shown in FIG. 6, the number of colonies fell by60% for cultures treated with 0.1 μM anti-nucleolin (A) whereas noeffect was found when cultures were treated with immunoglobulins of thesame isotype (B).

More especially, inhibition of the number of colonies with respect tothe control was also found for cultures treated with HB-19 and this in adose-dependent manner. A 59% decrease in the number of colonies incultures treated with 1 μM of HB-19 (FIG. 6C) was observed.

Similar results were obtained using murine melanomas such as B16-BL6 astarget cells. The results are presented in FIG. 7. Examination of theresults shows that both anti-nucleolin antibodies (A) and the moleculeHB-19 (B) inhibit the growth of B16-BL6 on wet agar in a dose-dependentmanner Inhibition in excess of 50% of the number of clones is observedin the presence of 1 μM of HB-19.

This set of results demonstrates that the compound HB-19 has aninhibitory effect on anchorage-independent cell growth. This is true ofboth cell models: human mammary carcinomas and mouse melanomas.

Effect of Pentavalent Compound HB19 on Angiogenesis Triggered byAngiogenic Factors

Given that surface nucleolin is present at the surface of activatedendothelial cells (9), the effect of HB-19 on the differentiation ofendothelial cells was tested.

Firstly, this effect was tested on the in vitro proliferation ofendothelial cells (HUVEC: Human umbilical vein endothelial cells). 20000HUVEC cells per well were cultured and compound HB19 was added indifferent concentrations on each day. Cells were counted after 6 days oftreatment. The results are given in FIG. 8A and show that, contrary tothe anti-nucleolin polyclonal antibody preparation used in the articleby Huang et al. (16), HB19 leads to inhibition of the proliferation ofendothelial cells.

The effect of the presence of HB19 (1 μM) on the differentiation ofHUVEC cells in a three-dimensional collagen gel cultured in the presenceof HARP angiogens (1 nM), VEGF (1 nM) and FGF-2 (3 nM) were also tested.After 4 days, tubular network structures were counted. The results arepresented in arbitrary units in FIG. 8B and show that HB19 also inhibitsthe differentiation of HUVEC cells in a three-dimensional collagen gelcultured in the presence of angiogenic factors.

Finally, the effect of HB-19 on the differentiation of endothelial cellswas tested in an in vivo angiogenesis model. This test mimics the firststages of the process leading to the formation of blood vessels.

This experimental model of angiogenesis consists in subcutaneouslyinjecting mice with matrigel containing the substance to be analysed forits angiogenesis stimulating or inhibiting properties. Matrigel wasremoved one week later, histological cuts were taken and the number ofendothelial cells (CD31+, factor VIII+) was quantified by image analysisafter immunohistochemistry. As can be seen in FIG. 8, HB-19 alone has noeffect on the recruitment of endothelial cells in matrigel. On the otherhand, it inhibits angiogenesis triggered by HARP or FGF-2.

Analysis of the results shows that HB-19 is capable of inhibitingangiogenesis triggered by proangiogenic factors such as FGF-2 or HARP.This shows a general angiostatic effect of HB-19 specifically targetingendothelial cells involved in angiogenesis.

Thus, compound HB19 drastically inhibits the proliferation anddifferentiation of HUVEC cells triggered by HARP, VEGF and FGF-2. Ittherefore has a much more pronounced effect than the anti-nucleolinmonoclonal antibodies used in the article by Huang et al. (16).

There are several advantages to using endothelial cells as ananti-cancer target cell. Contrary to tumour cells presenting geneticinstability, endothelial stable are extremely stable genetically, thuslimiting the mechanisms of resistance. In addition, since the moleculartarget is surface nucleolin, HB-19 principally targets activatedendothelial cells and therefore those that have entered theneo-angiogenesis phase. It should be noted that the tumoral endothelialcell divide 70 times more quickly than normal endothelial cells, whichis why tumoral endothelial cells are a major target, thus limitingpotential side effects.

In Vivo Anti-Tumour Effect of Pentavalent Compound HB19

The effect of pentavalent compound HB-19 on tumour growth in vivo wastested in a tumour growth model in athymic mice. In this experiment, thetarget cells originated from cancers of the human mammary glands:MDA-MB231.

Groups of 4 athymic mice (nude/nude) were injected in the flank with2×10⁶ cells. When tumour volume reached at least 200 mm³, mice wereeither treated or not by injection in the tumour (peritumoral orsubcutaneous route) of 100 μl every two days of a PBS solution (controlgroup) or HB-19 solution (5 mg/kg) or a commonly used clinical agent,tamoxifen (also called taxol, 10 mg/kg). Tumour size was measured ondays 7, 14, 21, 28, 34 and 40 using a calliper.

The results are presented in FIG. 8 and show that the peptide HB-19,used at a dose of 5 mg/kg, leads to inhibition of tumour growth withrespect to untreated control mice which had a ×7 larger tumour size.

Moreover, whereas tumour volume in mice treated with tamoxifen did notchange in a significant manner from the start of treatment, tumours inmice treated with HB-19 were undetectable after 21 days of treatment(FIG. 9A). Although tamoxfen at 10 mg/kg only leads to stabilisation oftumour volume or partial regression, pentavalent compound HB-19 at 5mg/kg actually leads to apparently total tumour regression.

In order to control these results, mice were sacrificed after 40 days oftreatment and tumours were removed then weighed. Average weight oftumours in untreated mice was 0.22 g (distribution 0.083-0.34 g), 0.06 gin those treated with tamoxifen 10 mg/kg (distribution 0.006-0.22 g) andno tumours were found in mice treated with HB-19 (FIGS. 9B and C), thusconfirming the estimation of tumour volume carried out by extracorporealmeasurement of size.

In another experiment, HB19 (5 mg/kg) was administered byintraperitoneal route (IP) and peritumoral route (SC) (FIG. 10). Theresults show that the anti-tumour action of HB-19 is equally effectiveby intraperitoneal route (IP) and peritumoral route, demonstrating notonly the unexpected efficacy of pentavalent compound HB-19 but also itsbioavailability in vivo at the tumour site, including in the case ofsystemic administration.

It should also be noted that in the course of treatment by HB-19, noabnormal physiological or behavioural sign was observed in treated mice.In addition, anatomical examination of the organs at the end of theexperiment did not reveal any visible sign of tissue toxicity nor anychange in blood formula or platelet count.

Moreover, it was not possible to detect HLA-DR⁺ human cells (thereforeMDA-MB231 tumour cells) in the peripheral blood cells of xenograftedmice treated with HB-19 (FIG. 11). In fact, contrary to untreated mice(PBS) in which the proportion of MDA-MB231 cells (HLA-DR⁺) represents22.2% of peripheral blood cells, this proportion is only 0.1% to 0.31%in the case of mice treated with HB-19 by subcutaneous orintraperitoneal route. These results suggest that HB-19 is also capableof preventing the circulation of tumour cells in the blood and,consequently, of preventing metastatic phenomena.

Conclusion

The results presented in Example 2 show that pentavalent compound HB-19is a powerful inhibitor of cell proliferation as a result of its dualtumour growth and angiogenesis effects. These observations have beenconfirmed in an in vivo model showing that treatment by peritumoralinjection of HB-19 is capable of inducing inhibition and even regressionof tumours in a PC3 cell xenograft model in athymic mice, and thiswithout tissue toxicity.

Compared to conventionally used treatments in cancer therapy, such astaxol, HB-19 in the experimental model used appears to be much moreefficacious. This greater efficacy of HB-19 compared to tamoxifen may bethe consequence of the inhibitory effect of HB-19 on both tumour growthand the formation of the vessels needed for tumour proliferation.Whether in the case of tumour cells or activated endothelial cells,HB-19 targets and blocks the proliferation of these two types of cells.

Many cells exist which have the property of inhibiting the proliferationof tumour cells. These molecules often have an effect without reallyhaving a cell target. In fact, for many chemotherapy agents, themolecular target present in a tumour cell is also found in normal cellswhich explains the many side effects of such treatments. Targetedbiological agents have fewer side effects as they block a target that isnot present or only minimally present in normal cells. Nucleolin presenton the surface of activated cells responds to this property andtherefore constitutes an ideal therapeutic target in the treatment ofcancer. It is also important to note that we target not only tumourcells but also activated endothelial cells. Moreover, surface nucleolindoes not appear to be restricted to a particular type of cancer.

The efficacy of dual targeting (tumour cells themselves and activatedendothelial cells) should be compared with the results of a studyconducted by Genentech which showed, in a randomized trial, that ananti-proliferative agent (5-fluorouracile) combined with ananti-angiogenic agent (Avastin) was highly effective in cases of humancolorectal cancer (28). In a phase III trial, Avastin treatment as acomplement to chemotherapy (irinotecan/5-fluorouracile/leucovorin)prolonged survival rates highly significantly by five months on average(20.3 months compared to 15.6 months) in individuals with previouslyuntreated metastatic colorectal cancer. In these patients, it was foundthat the length of time during which the tumour did not grow increasedfrom 6.2 months to 10.6 months compared to patients receiving onlychemotherapy (29).

This dual effect on tumour cells and endothelial cells makes HB-19 amolecule of choice in the treatment of cancer.

Anti-Tumour Activity of Trivalent Compound Nucant 01

Synthesis of Trivalent Compound Nucant 01

The chemical structure of trivalent compound Nucant 01 is given in FIG.2B. This compound has as a support a cyclic hexapeptide consisting ofalternate alanine (A) residues of configuration D and lysine residues(K) of configuration L. Three pseudopeptide units KΨPR (with Ψ=CH2-NH)are covalently bound to the ε amino group of each of the 3 lysineresidues (K).

The synthesis of compound Nucant 01 involves covalent coupling of theKΨPR unit to a C3-symmetric cyclic <<core>> molecule. Synthesis of thecore molecule is described by S. Fournel et al. (30). The protected KΨPRunit was assembled on a chlorotrityl type resin using a standard solidphase synthesis technique according to Fmoc type chemistry then cleavedfrom the resin under weak acid conditions. The protected KΨPR unit wasthen coupled to the epsilon NH2 group of each lysine residue (K) of thecore molecule on the basis of 1.1 KΨPR/1 cyclic molecule stoechiometry.Coupling was carried out in accordance with a BOP/HoBt activationprocedure for 48 hours. At the end of the reaction, the groupsprotecting KΨPR were cleaved in trifluoroacetic acid and the finalcompound precipitated in ether. The Nucant 01 molecule obtained at theend of the procedure was purified by HPLC and fully characterised bymass spectrometry.

Inhibitory Activity of Nucant 01 on the Proliferation of Tumour Cells inVitro

The effect of Nucant 01 on the proliferation of NIH-3T3 cells stimulatedby HARP was compared with that of pentavalent compound HB-19. QuiescentNIH-3T3 cells were stimulated or not by 4 nM of HARP in the presence ofHB19 or Nucant 01 at the concentrations indicated (0.1, 0.2, 0.4, 1, 2and 4 μM). After 24 hours of incubation, cell proliferation of NIH-3T3cells was determined by measuring the incorporation tritiated thymidineas described earlier.

Compared to the control stimulated by HARP in the absence of HB19 andNucant 01 (100% cell proliferation), it is found that the compoundNucant 01, which is only trivalent for KPR units, also leads to 50%inhibition of cell proliferation of NIH-3T3 cells stimulated HARP at aconcentration of 2 μM. This result therefore demonstrates that syntheticmultivalent compounds with at least 3 pseudopeptide units of formula (I)grafted on a cyclic peptide also make it possible, in the same way aspentavalent compound HB-19 whose support is a linear peptide, to inhibittumour cell proliferation triggered by HARP.

The required concentration is 10 times higher than that needed to obtainthe same level of inhibition with HB-19 (0.2 μM) but the compound Nucant01 only has 3 pseudopeptide units of formula (I) whereas compound HB-19has 5.

It is therefore probable that increasing the number of pseudopeptideunits of formula (I) grafted on a cyclic peptide of the kind used inNucant 01, to 4 or 5 would further increase the efficacy of thecompound, possibly up to 100 times.

Conclusion

These results clearly demonstrate the importance of a multivalent formof the pseudopeptides of formula (I) for the activity of the compoundsused in the invention, with the possibility of using a variety ofsupports, and linear peptides or cyclic peptides can be used withoutaffecting the efficacy of the compounds.

Other acceptable supports can therefore be used equally well as long asthey allow at least 3, preferably 3 to 8, preferably 4 to 6, preferably5 or 6 pseudopeptide units of formula (I) to be grafted on.

Anti-Tumour Activity of Pentavalent Compounds Nucant 2 and Nucant 3

Synthesis of Pentavalent Compounds Nucant 2 and Nucant 3

Two peptide supports known to adopt a helicoidal structure on which KΨPRunits were anchored were assembled by solid phase synthesis. Thesesupports were made up of repeated units of sequence Aib-Lys-Aib-Gly forNUCANT 2 and Lys-Aib-Gly for NUCANT 3 linked together five times, inwhich Aib represents 2-amino-isobutyric acid. Assembly was carried outby means of Boc type chemistry. Protective Fmoc groups of the lysineresidue side chain were then cleaved by treatment with piperidine (3times 5 minutes) in DMF. The five εNH2 groups of lysine then acted asanchors for the KΨPR units (with Ψ=CH₂—N). Final acid cleavage wascarried out in hydrofluoric acid. After precipitation of peptides inether, dissolution in aqueous conditions and freeze-drying, NUCANT 2 andNUCANT 3 analogues were purified by HPLC and analysed by massspectrometry then freeze dried.

Inhibitory Activity of Nucant 2 and Nucant 3 on the Proliferation ofTumour Cells in Vitro

The effect of Nucant 2 and Nucant 3 on the proliferation NIH-3T3 cellstriggered by HARP was compared to that of pentavalent compound HB-19.Quiescent NIH-3T3 cells were stimulated or not by 4 nM by HARP in thepresence of HB 19, Nucant 2, or Nucant 3 at the concentrations indicated(0.1, 0.25 and 0.5 μM). After 24 hours of incubation, cell proliferationof NIH-3T3 cells was determined by measuring the incorporation tritiatedthymidine as described earlier.

In this experiment, HB19 inhibits with IC50 at 0.1 μM. Nucant 2 andNucant 3 inhibit cell proliferation in a dose-dependent manner with IC50at 1.5 μM.

The pentavalent compounds Nucant 2 and Nucant 3 are therefore capable ofinhibiting cell proliferation in a dose-dependent manner, with a IC50(concentration leading to 50% inhibition of cell proliferation) verysimilar to that of compound HB19.

The effect of Nucant 3 on the proliferation of NIH-3T3 cells treated by5% of FCS was also compared to that of compound HB-19. NIH-3T3 cellsmade quiescent by serum deprivation were stimulated by 5 FCS in thepresence or not of different concentrations of NUCANT 3, 6 or 7 rangingfrom 0.125 to 2 μM. After 24 hours in incubation, cell proliferation isdetermined by measuring the incorporation of tritiated thymidine asdescribed earlier.

The results are presented in FIG. 14 as a percentage with respect tocontrol cells stimulated by 5 FCS. The results show that NUCANT 3 has aninhibitory effect on cell proliferation. Analysis of the graph showsthat NUCANT 3 has an ID₅₀ value (Inhibitory Dose at 50%) that is similarbut slightly lower than that of the HB-19 molecule and is thereforeslightly less effective than HB-19. No effect was observed with NUCANT 3on non-stimulated cells, indicating the absence of any toxicity of thesemolecules.

Conclusion

These results also demonstrate the importance of a multivalent form ofpseudopeptides of formula (I) for the activity of the compounds used inthe invention, the support used equally well being a linear peptidewhich contains or not structural elements (β fold, β sheets), a linearpeptide with a helicoidal structure or even a cyclic compound, withoutthis affecting the efficacy of the structure.

Various acceptable supports can thus be used interchangeably as long asthey allow at least 3, preferably between 3 and 8, preferably between 4and 6, preferably 5 or 6 pseudopeptide units of formula (I) to begrafted on.

Anti-Tumour Activity of Hexavalent Compounds Nucant 6 and Nucant 7

Synthesis of Hexavalent Compounds Nucant 6 and Nucant 7

These hexavalent compounds were obtained using the same synthesisprocess as that used for pentavalent compounds Nucant 2 and 3.

Inhibitory Activity of Nucant 6 and Nucant 7 on the Proliferation ofTumour Cells in Vitro

The effect of hexavalent compounds Nucant 6 and Nucant 7 on theproliferation of NIH-3T3 cells triggered by 5% FCS was compared withthat of pentavalent compound HB-19. NIH-3T3 cells made quiescent byserum deprivation stimulated by 5% FCS in the presence of variousconcentrations of NUCANT 3, 6 or 7 ranging from 0.125 to 2 μM. After 24hours of incubation, cell proliferation was determined by measuring theincorporation of tritiated thymidine as described above.

The results are given in FIG. 14 as a percentage with respect to controlcells stimulated by 5 FCS. The results show that NUCANT 6 and 7 have aninhibitory effect on cell proliferation. NUCANT 6 and 7 have an ID₅₀value (Inhibitory Dose at 50%) that is similar but slightly lower thanthat of the HB-19 molecule and are therefore slightly less effectivethan HB-19. No effect was observed with NUCANT 6 or 7 on non-stimulatedcells, indicating the absence of any toxicity of these molecules.

Conclusion

These results are a further demonstration of the importance of amultivalent form of pseudopeptides of formula (I) for the activity ofthe compounds used in the invention, the support used equally well beinga linear peptide which contains or not structural elements (β bend, βsheets), a linear peptide with a helicoidal structure or even a cycliccompound, without this affecting the efficacy of the construction.

In particular, the form with 6 pseudopeptide units appears to be highlyeffective in obtaining the desired anti-proliferation andanti-angiogenic effects.

Nucant 6 and Nucant 7 are More Powerful Inhibitors of Surface Nucleolinthan HB-19

Nucant 6 and Nucant 7 are More Powerful Inhibitors than HB-19 andInhibit the Activity of Surface Nucleolin

The activity of surface nucleolin was tested in HeLa P4 cells using thetechnique we have described previously (13).

The results presented in FIG. 15 show that inhibition of surfacenucleolin activity by Nucant 3 is comparable to that of HB-19. On theother hand, Nucant 6 and Nucant 7 have greater surface anti-nucleolinactivity than HB-19. HB-19 and Nucant 3 have an ID₅₀ value (InhibitoryDose at 50%) which is between 0.1-0.2 μM, whereas Nucant 6 and Nucant 7have an ID₅₀ value below 0.1 μM. Moreover, Nucant 6 and Nucant 7 used at0.8 μM lead to over 95% inhibition of surface nucleolin activity.

HB-19, Nucant 3, 6, and 7 Lead to Inhibition of Surface Nucleolin inHuman Breast Cancer Cells, MDA-MB 231

Surface nucleolin plays an important role in proliferation and tumourangiogenesis. HB-19 as well as related molecules Nucant 3, Nucant 6 andNucant 7 specifically bind to surface nucleolin, thus blocking tumourgrowth and angiogenesis. After these pseudopeptides bind to surfacenucleolin, the [pseudopeptide-nucleolin] complex is rapidly internalisedby means of an active process. These results are presented in FIG. 16Aand show that treatment of cells with HB-19 (line 1), Nucant 3 (line 2),Nucant 6 (line 3) or Nucant 7 (line 4), result in a reduction in thepresence of surface nucleolin compared to untreated cells. Thisreduction is observed after 24 hours of treatment but also after 48hours of treatment in which case nucleolin becomes non-detectable.

It is interesting to note that there is a considerably greater reductionin surface nucleolin when cells are treated for 24 hours withpseudopeptides Nucant 6 and Nucant 7 (lines 3 and 4) compared to cellstreated with HB-19 and Nucant 3 (lines 1 and 2). The same observation ismade after 48 hours of treatment. It is important to note that thereduction in surface nucleolin is not the result of a reduction in theintracellular amount of surface nucleolin. In fact, identical amounts ofnucleolin are found in cell extracts of cells either treated or not byHB-19 or by the various types of Nucant (FIG. 16B). Similarly way, it isfound that there is no difference between the electrophoresis profilesof proteins extracted from cells whether treated or not by HB-19 or thedifferent types of Nucant. This result illustrates that proteinsynthesis is not affected by HB-19 or the different types of Nucantstudies. Moreover, it is interesting to note that HB-19 and thedifferent types of Nucant studied have no cytotoxic effect that mightexplain the observed reduction in surface nucleolin.

Conclusions

This set of results shows that:

-   -   a) nucleolin is expressed in large quantities at the surface of        tumour cells, for example in human breast cancer cells        (MDA-MB231).    -   b) treatment by HB-19, Nucant 3, Nucant 6 and Nucant 7 triggers        a marked decrease in the nucleolin <<pool>> present at the        surface of cells.    -   c) the pseudopeptides Nucant 6 and Nucant 7 are more effective        in producing a reduction in the nucleolin <<pool>> present at        the surface of cells, as well as in inhibiting the activity of        surface nucleolin.

Effect of HB-19 and Nucant 7 on Angiogenesis

Methods

The effect of HB-19 and Nucant 7 on angiogenesis was tested in an exvivo model of angiogenesis, the chorioallantodoin membrane (CAM) ofchicken embryo.

20 μl of water containing or not (control) HB-19 (10 μM; 0.6 μg) orNucant 7 (10 μM; 0.8 μg) are deposited on the surface of CAM.Observation of vessels was carried out after 48 h of incubation

Results

The results are presented in FIG. 17 and show that after 48 hours ofincubation, HB-19 and Nucant 7 clearly lead to inhibition ofangiogenesis. An image analysis study, taking into consideration notonly capillary length but also the number of branches, suggestsinhibition in the region of 50% compared to the control.

Anti-Inflammatory Activity of Compounds HB 19 and Nucant 7

Inhibition by HB-19 of the Production TNF-α by Human Primary PBMCStimulated by LPS.

Methods

PBMCs were isolated by centrifugation on a Ficoll density gradient usingwhole human blood EDTA-potassium and resuspended in RPMI 1640 containing1% human serum AB (Invitrogen). Cells at a concentration of 10⁶ cell/0.5ml, in the absence (0) or presence (1 and 5 μM) of HB-19, werestimulated with 100 ng/ml of LPS from Escherichia coli type 0111: B4 and055: B5, and LPS from Salmonella enterica serotype Re 595. The samePBMCs were stimulated with PMA: Ionomycin (Phorbol 12-myristate13-acetate: Ionomycin) at 20 ng/ml: 1 μM. PBMC cultures were incubatedat 37° C. in an incubator with 5% CO₂. The level of TNF-α protein wasmeasured by ELISA in culture supernatants collected after 20 hours ofincubation.

Results

The results (see FIG. 18) show that freshly isolated PBMC produces TNF-αand that this constitutive production of TNF-α is not affected by HB-19.On the other hand, HB-19 inhibits the production of TNF-α by PBMC in adose-dependent manner in response to stimulation by various LPSpreparations from Escherichia coli or Salmonella enterica. This effectis specific because HB-19 does not have an effect on the production ofTNF-α by PBMC in response to stimulation by PMA-Ionomycin.

At 5 μM of HB-19, the production TNF-α by human PBMC in response tostimulation by various LPS preparations is inhibited to a similar degreeto the base level observed in the absence of stimulation by LPS.

Inhibition by HB-19 and Nucant 7 of the Production of TNF-α and IL-6 byMurine Macrophages from Primary Peritoneum Stimulated by LPS

Method

In order to obtain stimulated macrophages, 7 to 8 week old balb/c micereceived and intra-peritoneal injection 4 days prior to the experimentusing 1.5 ml of a thioglycolate solution (3% saline solution).Macrophages were collected in the peritoneal cavity by washing theperitoneum with 5 ml of RPMI medium containing 1% foetal calf serum.Cells were then placed on plates at a density of 10⁶ cells/0.5 ml inRPMI 1640 medium, incubated at 37° C. in an incubator with 5% CO₂, andnon-adhering cells were removed 2 hours later.

Macrophages, in the absence (−) or presence (+) of 4 μM HB-19 or 10 μMNucant 7, were either non-stimulated or stimulated with LPS fromEscherichia Coli serotype 0111: B4 at 10 ng/ml, 100 ng/ml and 1000ng/ml. Cell cultures were incubated at 37° C. in an incubator with 5%CO₂ for 20 hours. TNF-α and IL-6 protein levels were measured by ELISA.

Results

The results obtained for HB-19 are presented in FIG. 19. At 4 μM ofHB-19, the production of TNF-α and IL-6 by murine peritoneal macrophagesin response to stimulation by LPS is significantly inhibited. If thebase level observed in the absence of stimulation by LPS is taken intoaccount, the net degree of inhibition is 72-75% for TNF-α and 68-71% forIL-6.

The results obtained with Nucant 7 are presented in FIG. 20. At 10 μM ofNucant 7, the production of TNF-α and IL-6 by murine peritonealmacrophages in response to stimulation by LPS is almost completelyinhibited because the cytokine production level observed in culturestreated with Nucant 7 in response to stimulation by LPS is similar tothat observed in the absence of LPS.

If the base level observed in the absence of stimulation by LPS is takeninto account, the degree of inhibition at 10 μM of Nucant 7 is over 95%in cultures stimulated by 10, 100 and 1000 ng/ml of LPS. The fact thatthe degree of inhibition is not altered in the presence of a 100 timesgreater LPS concentration suggests that the mechanism of inhibition byNucant 7 is principally due to binding to surface nucleolin. In fact, ifthe mechanism of inhibition by Nucant 7 were the result of interactionwith LPS, the inhibitory effect would be lower at 100 ng/ml of LPS than10 ng/ml of LPS.

Inhibition by HB-19 of the Production of IL-8 and Expression of ICAM-1by HUVEC Cells Stimulated by LPS.

Method

HUVEC cells at a concentration of 10 000 cells/cm² were cultured in96-well plates in EBM-2 medium containing 2% foetal calf serum. Cells inthe absence or presence of 5 μM of HB-19 were stimulated by LPS fromEscherichia coli serotype 055: B5 at 100 ng/ml. Cell cultures wereincubated at 37° C. in an incubator with 5% CO₂ for 20 hours. Levels ofIL-8 and ICAM-1 were measured by ELISA. HUVEC cells in the absence orpresence of 5 μm of HB-19 were used as a control to show the base level.

Results

If the base level observed in the absence of stimulation by LPS is takeninto account, the degree of inhibition of IL-8 and ICAM-1 production at5 μM of HB-19 is around 50%. These results therefore demonstrate thepotential efficacy of HB-19 and related compounds of the Nucant type asinhibitors of the production of IL-8 and ICAM-1 (see FIG. 21).

Inhibition by HB-19 of the Production TNF-α by Human Primary PBMCStimulated by Inactivated Staphylococcus Aureus Bacteria

Infection by Staphylococcus aureus has been shown to be one of the majorcauses in endocarditis (24, 25).

The inventors therefore measured TNF-α and IL-6 levels in human primaryPBMC cultures in response to Staphylococcus aureus bacteria inactivatedby heat (HKSA, <<heat-killed Staphylococcus aureus>>), in the absence(control) or presence of 10 μM of the compounds HB-19, Nucant 3, Nucant6, or Nucant 7. As the positive control (Dex.), PBMC was also treatedwith dexamethasone which is a glucocorticosteroid with knownanti-inflammatory and immunosuppressant activity.

Method

PBMC was isolated by centrifugation on a Ficoll density gradient usingwhole human blood EDTA-potassium and resuspended in RPMI 1640 mediumcontaining 1% human serum AB (Invitrogen). Cells at a concentration of10⁶ cells/0.5 ml in the absence (Control) or presence (10 μM) of HB-19,Nucant 3, Nucant 6, Nucant 7, and dexamethasone (1 μg/ml) werestimulated with 10⁸ HKSA/ml particles (InvivoGen, San Diego, USA). PBMCcultures were incubated at 37° C. in an incubator with 5% CO₂. The levelof TNF-α and IL-6 protein was measured by ELISA in culture supernatantscollected after 20 hours of incubation.

Results

The results are presented in FIG. 22 and show that all thepseudopeptides tested (HB-19, Nucant 3, Nucant 6, and Nucant 7) make itpossible to obtain significant inhibition of TNF-α and IL-6 productionin response to stimulation by heat killed Staphylococcus aureusbacteria. The pseudopeptides are as effective as standardanti-inflammatory treatment such as dexamethasone.

Thus the pseudopeptides of formula (I) such as HB-19, Nucant 3, Nucant 6and Nucant 7, can also be used as treatment for cardiac inflammation,particularly in the treatment of endocarditis of infectious origin.

Conclusion

The results obtained by the inventors therefore show that the compoundsHB-19 and Nucant 7 are capable of inhibiting the production of bothproinflammatory cytokines such as TNF-α and IL-6, and the production ofchemokine IL-8 and adhesion molecule ICAM-1 by various cell types inresponse to stimulation by LPS.

Moreover, these compounds also lead to significant inhibition of theproduction of production proinflammatory cytokines such as TNF-α andIL-6 in response to stimulation by Staphylococcus aureus bacteria, oneof the agents responsible for numerous forms of endocarditis ofinfectious origin.

Consequently, these compounds as well as the related compounds offormula (I) described in this invention are capable of inhibiting theproduction of proinflammatory cytokines and molecules involved in therecruitment of leukocytes to the site of inflammation. These compoundscan thus be used in anti-inflammatory applications, notably in thetreatment of the various diseases mentioned in the general description.

1. A polyvalent synthetic compound comprising a support on which atleast 3 pseudopeptide units are grafted, said compound being of formula(I):

wherein the support is a linear peptide, and wherein grafted supportresidues are separated by at least one amino acid; each X independentlyrepresents any amino acid; Y₁ and Y₂ are selected independently fromarginine (R) and lysine (K); Z is proline; n and i are independently 0or 1; m is an integer between 0 and 1; k is an integer between 3 and 8;and Ψ represents a modified peptide bond selected from the groupconsisting of a reduced bond (—CH₂NH—), a retro-inverso bond (—NHCO—), amethyleneoxy bond (—CH₂—O—), a thiomethylene bond (—CH₂—S—), a carbabond (—CH₂CH₂—), a ketomethylene bond (—CO—CH₂—), a hydroxyethylene bond(—CHOH—CH₂—), an E-alkene bond or a (—CH═CH—) bond.
 2. The polyvalentsynthetic compound of claim 1, wherein the support comprises the aminoacid sequence of KX₁KX₄KX₁K, where X₁ is optional and selected fromlysine (K), valine (V), alanine (A), glutamic acid (E) and isoleucine(I), and X₄ is optional and selected from valine (V), alanine (A),glutamic acid (E) and isoleucine (I).
 3. The polyvalent syntheticcompound of claim 1, wherein the support is selected from a linearpeptoid, or linear foldamer.
 4. The polyvalent synthetic compound ofclaim 1, wherein the support is a linear peptide consisting of an aminoacid sequence selected from SEQ ID NO :1, SEQ ID NO :2, SEQ ID NO :3,SEQ ID NO :4, SEQ ID NO :8, SEQ ID NO :9, SEQ ID NO :13, SEQ ID NO :14,SEQ ID NO :15, SEQ ID NO :18, or SEQ ID NO :19.
 5. The polyvalentsynthetic compound of claim 1, wherein the compound is selected from thefollowing compounds:


6. The polyvalent synthetic compound of claim 1, having a support as setforth in SEQ ID NO :17.
 7. The polyvalent synthetic compound of claim 1,wherein said pseudopeptide units are grafted directly on said support.8. The polyvalent synthetic compound of claim 1, wherein saidpseudopeptide units are grafted on said support by means of a spacer. 9.A pharmaceutical composition comprising an effective amount of apolyvalent synthetic compound according to claim 1 and an excipient. 10.Method for treating inflammatory diseases comprising administering aneffective amount of a polyvalent synthetic compound as defined in claim1 to a patient in need thereof, wherein the compound is effective intreating, alleviating, or ameliorating the inflammatory disease.
 11. Themethod according to claim 10, wherein said inflammatory disease isselected from autoimmune diseases, septicaemia, septic shock, cardiacinflammatory diseases, graft rejection, trauma, inflammatory diseases ofthe joints, inflammatory diseases of the gastrointestinal system,inflammatory diseases of the skin, inflammatory diseases of therespiratory system and allergies.
 12. The method according to claim 11,wherein said inflammatory disease is an autoimmune disease.
 13. Themethod according to claim 12, wherein said autoimmune disease is lupusor rheumatoid polyarthritis.
 14. The method according to claim 11,wherein said inflammatory disease is septic shock.
 15. The methodaccording to claim 11, wherein said inflammatory disease isendocarditis.